Correction to "The kinase domains of obscurin interact with intercellular adhesion proteins"

Abstract

Hu, L.-Y.R. and Kontrogianni-Konstantopoulos, A. (2013), The kinase domains of obscurin interact with intercellular adhesion proteins. The FASEB Journal, 27: 2001-2012. https://doi.org/10.1096/fj.12-221317 The journal was informed of possible duplicated areas in portions of lanes 3-5 in Figure 2G, which include negative controls and therefore contain no signal. The authors provided replicate original autoradiograms showing similar findings to the published Figure 2G. The conclusions of Figure 2G and the study did not change. The journal was informed of possible similarities in Sypro Ruby stained lane 2 of Figures 2E and 4D, labeled "N.I. Lysates +/Purified SK2 -" and "N.I. Lysates +/Purified SK1 -", respectively, which are negative controls and therefore contain no signal. The authors were not able to retrieve the original Sypro Ruby stained images shown in Figures 2E and 4D but provided Coomassie Blue (Figure 2E) and Western blotting (Figure 4D) assays of the relevant samples showing the same results. The conclusions of Figures 2E, 4D, and the entire study did not change. The corrected Figure 2 is as follows: FIGURE 2. N-cadherin is a substrate of SK2. (A, B) Schematic diagrams illustrating the SK2 bait (A) and N-cadherin prey (B) constructs. αC, α-helix C; AS, activation segment; CDR, extracellular cadherin repeats; CL, catalytic loop; I, intracellular domain; NB, nucleotide binding domain; PRO, prodomain; TM, transmembrane region. (C) Identification of minimal interacting domains using the Y2H system; relative strength of the identified interactions is indicated by + and - symbols. (D, D') Fluorescent (D) and bright field (D') images obtained from the PLA assay. Scale bar = 30 μm. (E) Purified His-tagged SK2 protein analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue-R250. (F) Purified control GST-protein and recombinant N-cadherin peptides fused to GST separated by SDS-PAGE and stained with Coomassie Brilliant Blue-R250. (G) Autoradiogram of an in vitro kinase assay using His-tagged SK2 and different substrates; bands denoted as P-SK2 and PP-SK2 represent SK2 species autophosphorylated to different extents. FIGURE 4. A small obscurin kinase isoform undergoes glycosylation. (A-C) Adult mouse heart lysates were incubated with lectin resins. Eluted fractions were immunoprobed for the presence of giant obscurin-B (A) or smaller obscurin kinase isoforms prior to (B, top panel) and after (C) treatment with PNGase F glycosidase. Protein lysates were also treated with PNGase F before they were applied to the lectin resin (B, bottom panel); ME and SE, elution buffers (see Materials and Methods), El1 and El2, eluted fractions 1 and 2. (D) Purified His-tagged SK1 protein analyzed by SDS-PAGE and Western blotting (WB) assay probed with the ObscKin3 antibody. (E) Purified control GST-protein and recombinant NKA 1 peptide fused to GST, separated by SDS-PAGE, and stained with Coomassie Brilliant Blue-R250. (F) Autoradiogram of an in vitro kinase assay using His-tagged SK1 and different substrates; bands marked as P-SK1 represent autophosphorylated species of SK1. Notably, the autophosphorylation activity of SK1 is significantly diminished in the presence of recombinant NKA 1, possibly due to direct binding of the latter to the catalytic part of the former, as our Y2H results indicated.