Published Erratum
doi: 10.1371/journal.pbio.1002414.
eCollection 2016 Mar.
Correction: HER2 Phosphorylation Is Maintained by a PKB Negative Feedback Loop in Response to Anti-HER2 Herceptin in Breast Cancer
- PMID: 26949965
- PMCID: PMC4780807
- DOI: 10.1371/journal.pbio.1002414
Item in Clipboard
Published Erratum
Correction: HER2 Phosphorylation Is Maintained by a PKB Negative Feedback Loop in Response to Anti-HER2 Herceptin in Breast Cancer
PLoS Biol.
.
No abstract available
Figures
(A) SKBR3 cells were lysed for Western blot analysis after pre-treatment with 40 μg/ml Herceptin for a period of 4 h, 2 d, or 10 d. Equal amounts of protein were loaded in each lane, and multiple parallel SDS-PAGE gels were run. Membranes were blotted with appropriate antibodies to assess the indicated proteins (shown here and Fig 2E). Note that only one representative actin control is shown for this experiment. (B) SKBR3 cells were grown in 24-well plates and left to grow for at least 24 h before being treated with 40 μg/ml Herceptin for different durations, as illustrated. The viable cells were counted in a cell viability analyzer using trypan blue to stain the dead cells. (C) SKBR3 cells were incubated with either donor alone (HER2-Cy3b) or donor and acceptor (HER2-Cy3b+pHER2-Cy5) to assess HER2 phosphorylation by FRET after pre-treatment with 40 μg/ml Herceptin for different durations, as illustrated. (D) BT474 cells were lysed for Western blot analysis after pre-treatment with 40 μg/ml Herceptin for a period of 1 h, 1 d, or more than 8 months (m) with replacement every week. Equal amounts of protein were loaded in each lane. Membranes were blotted with antibodies for total and phosphorylated HER2 and actin. (E) BT474 cells were lysed for western blot analysis for the indicated proteins after 40 μg/ml Herceptin treatment for 1 hour. Note that other treatment conditions were assessed at the same time in other lanes of the same gel but they were cut from the membranes as they were irrelevant for these blots. The images were spliced from the same gel with a solid line is included to indicate the splice. (F) SKBR3 cells were lysed after 40 μg/ml Herceptin treatment or stimulation with heregulin 100ng/ml for the indicated durations. Equal amounts were loaded on an SDS gel, and membranes were probed for the indicated proteins.
(A) In the left two panels, BT474 cells were immunoprecipitated (IP) with either intracellular anti-EGFR or anti-HER2 antibody after 1 h of 40 μg/ml Herceptin treatment. Following the immunoprecipitation, the cell lysate was loaded unto an SDS gel, and a Western blot analysis was performed. The membrane was probed with either anti-EGFR or anti-HER2 antibody, as illustrated. The cell lysate from those immunoprecipitated with anti-HER2 antibody, was also probed for pEGFR shown in Fig 2B upper second panel. In the right panel, BT474 cells treated for 1 h with 40 μg/ml Herceptin were lysed, and equal amounts were loaded on a gel. Membrane was probed for phosphorylated ERK and actin. (B) In the left upper and lower panels, BT474 and SKBR3 cells were immunoprecipitated with biotinylated anti-HER2 antibody after the cells were treated for 1 h with 40 μg/ml Herceptin. Following the immunoprecipitation, equal amounts of the cell lysates were loaded unto an SDS gel, a Western blot analysis was performed, and the membrane was probed with anti-HER3 antibody. In the top right two panels, BT474 cells were immunoprecipitated with biotinylated anti-HER2 antibody after 1 h of Herceptin treatment. Western blot analysis was done using antibodies that recognised pEGFR and pHER3. Note that pEGFR blot was from the same experiment using the same cell lysate immunoprecipitated with anti-HER2 antibody in Fig 2A as described above. In the lower right three panels, BT474 cells were treated with 40 μg/ml Herceptin for 1 h before the lysate was immunoprecipitated using the anti-HER2 antibody Herceptin. After immunoprecipitation, lysate was loaded onto an SDS gel. A Western blot analysis was performed, and the membrane was probed with anti-pEGFR, anti-pHER3, or anti-pHER4 antibodies. Note that the pEGFR and pHER4 blots were from the same experiment using the same cell lysate immunoprecipitated with Herceptin however the pHER3 blot was from a different experiment. (C) BT474 cells were treated for 1 h with 40 μg/ml Herceptin, and the cells were lysed using Hepes buffer and homogenised before analysing the levels of heregulin and betacellulin by ELISA. (D) In the left panel, serum-free medium of untreated and Herceptin-treated SKBR3 cells was immunoprecipitated for heregulin. A Western blot analysis was then performed, and the membrane was probed for heregulin. In the right panel the same technique was used to look at betacellulin levels in the medium of SKBR3 cells. (E) SKBR3 cells were lysed for Western blot analysis after pre-treatment with 40 μg/ml Herceptin as described in Fig 1A. An equal amount of protein was loaded in each lane. Multiple parallel SDS-PAGE gels were run, and the membranes were cut in different parts according to molecular weight to analyse the indicated proteins using the appropriate antibodies. Only one representative actin control was shown for this experiment. (F) BT474 cells were lysed after pre-treatment with 40 μg/ml Herceptin for 1 h, 4 h, or 8 m. Equal amounts were loaded on an SDS gel, and membranes were probed for phosphorylated HER3 and actin. Note that we also had treatment conditions related to Herceptin withdrawal on the same gel but these were cut from the gel for clarity and the images were spliced from the same gel. A solid line is now included to indicate the splice. (G) BT474 cells were treated for 5 d with either 40 μg/ml Herceptin alone or concurrent 40 μg/ml Herceptin treatment with exogenous 100 ng/ml EGF, betacellulin (BTC), or heregulin (HRG) stimulation. Cells were then counted using a cell counter to assess cell viability. For statistical analysis, untreated and Herceptin-treated samples were compared using the Mann-Whitney test. The Herceptin-treated samples were then compared with samples treated with Herceptin and concurrent growth factor stimulation.
(A) SKBR3 cells were transfected with siRNA against ADAM17 or a control sequence. Three days after transfection, cells were treated with 40 μg/ml Herceptin for 1 h. Lysates were loaded on an SDS gel. The membrane was probed for the indicated proteins (left panel and Fig 5C). The cells were also lysed in Hepes buffer for heregulin detection using ELISA (right panel). (B) SKBR3 cells were treated with 40 μg/ml Herceptin for 1 h, and ADAM17 mRNA levels were studied by QPCR. The Mann-Whitney test was performed to determine statistical significance of the up-regulation of ADAM17 mRNA after Herceptin treatment. (C) BT474 cells (left two panels) and SKBR3 cells (right two panels) were treated with an increasing dose of Herceptin over a period of 1 h. Cell lysate was loaded onto an SDS gel, and a Western blot analysis was performed. The membrane was probed with anti-ADAM17 and anti-pPKB antibodies. Quantification of three separate experiments is depicted in graphs for pPKB and ADAM17; representative blots are shown above the graphs.
(A) BT474 cells were treated for 1 h with 40 μg/ml Herceptin (Herc), 2.5 μM PKB inhibitor (PKB inh), 40 μg/ml IgG control, or DMSO control at 37°C. After 1 h, cells were lysed and analysed for total and phosphorylated PKB levels using MSD multiplex kits. (B) BT474 cells were treated with the indicated treatment conditions for 1 h and the cell lysates were loaded on an SDS gel, and the membranes were probed for the indicated proteins. (C) BT474 cells pre-treated with PKB inhibitor were lysed in Hepes buffer and homogenised before analysing protein levels of heregulin using ELISA. (D) BT474 cells were treated with PKB inhibitor for 1 h, and ADAM17 mRNA levels were studied by QPCR. Statistical significance was determined using the Mann-Whitney test.
(A) SKBR3 cells were treated with 10 μM ADAM17 inhibitor (inh), 10 μM ADAM10/17 inhibitor, 10 μM ADAM inhibitor TAPI-1, or a combination of TAPI-1 and 40 μg/ml Herceptin (Herc) for 1 h. Cells were then lysed, and equal amounts of protein were loaded on an SDS gel. Membrane was probed for phosphorylated HER2 and actin. (B) FRET experiments to assess HER2 phosphorylation in SKBR3 cells. The cells were treated with 40 μg/ml Herceptin for 1 h with or without TAPI-1. The medians of the lifetimes were compared with the basal condition using the Mann-Whitney test. (C) Same experiment as Fig 3A; the membranes were probed for the indicated proteins. Only one representative actin control was shown for this experiment. (D) BT474 cells were grown in 24-well plates and left to grow for at least 24 h before being treated for different durations with 40 μg/ml Herceptin, 10 μM TAPI-1, 10 μM ADAM17 inhibitor, or a combination of ADAM inhibitors with Herceptin, as illustrated. The viable cells were counted using a cell counter.
Erratum for
-
HER2 phosphorylation is maintained by a PKB negative feedback loop in response to anti-HER2 herceptin in breast cancer.PLoS Biol. 2010 Dec 21;8(12):e1000563. doi: 10.1371/journal.pbio.1000563. PLoS Biol. 2010. PMID: 21203579 Free PMC article.
References
-
- Gijsen M, King P, Perera T, Parker PJ, Harris AL, Larijani B, et al. (2010) HER2 phosphorylation is maintained by a PKB negative feedback loop in response to anti-HER2 herceptin in breast cancer. PLoS Biol. 8(12): e1000563 doi:10.1371/journal.pbio.1000563 - DOI - PMC - PubMed
Publication types
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources
Research Materials
Miscellaneous
