The In-Cell Western immunofluorescence assay to monitor PROTAC mediated protein degradation

Abstract

The In-Cell Western plate-based immunofluorescence assay is a useful methodology for monitoring protein levels and provides a facile moderate through-put method for PROTAC and degrader optimization. The method is compared to other reported assays used for PROTAC development. The advantages of this method are the greater through-put compared to Western blots due to its plate-based method and the ease to transfer between cells lines. Adherent cell lines are preferred, although suspension cells can be used following recommended modifications and precautions to the protocol. This method requires a high-quality antibody that recognizes the protein epitope in its cellular context, and in general provides data similar to Western blots with higher assay through-put.

Keywords: Degrader; In-Cell Western; Lead optimization; PROTAC; Protein degradation.

Fig. 1

The plate map for seeding SK-MEL-2 cells. Out-lying wells are not used for analysis due to edge effects and are filled with PBS to help avoid evaporation.

Fig. 2

The plate map for the primary and secondary antibody dilution. The out-lying wells are not used for the assay.

Fig. 3

Screen shot of LI-COR Image Studio Software^® image under the In-Cell Western protocol.

Fig. 4

In-Cell Western analysis normalized to CellTag 700.

Fig. 5

In-Cell Western analysis to define the well types.

Fig. 6

In-Cell Western analysis under the Grid Sheet tab to define the Qualification data to export to an Excel file.

Fig. 7

Signal Linearity. (A) In-Cell Western Analysis to show the signal linearity between the fluorescent signal from the secondary antibody IRDye 800CW to the cell number normalized to CellTag 700 stain. (B) The partial graph of signal linearity is used to calculate a more accurate R value.

Fig. 8

(A) Signal linearity for CellTag 700 stain at various cell densities without addition of secondary antibody IRDye 800CW. (B) Signal to Noise ratio calculated for various primary antibody dilutions and cell number.

Fig. 9

(A) Plate Image for visualization of CDK6 with secondary antibody IRDye 800CW. (B) Plate Image for visualization with CellTag 700 stain. (C) Plate Image A and B merged.

Fig. 10

Plate Map for ICW Analysis.

Fig. 11

Plate Map for Compound Treatment, Primary and secondary Antibody.

Fig. 12

Western Blot for CDK6 in SK-MEL-2 cells.

Fig. 13

ICW Analysis of CDK6 levels after pharmacological treatment. Treatment of nine compounds monitoring CDK6 protein levels at 9 different concentrations. (A) Compounds tested starting from 10μM include AC-03-092, a resynthesis of BSJ-03-123 used as a reference compound and Palbociclib. (B) Compounds tested starting from 1μM include YX-2-107 a reported CDK6 degrader.

Fig. 14

ICW Analysis of Live SK-MEL-2 cells after pharmacological treatment. Treatment of nine compounds monitoring SK-MEL-2 cell number at 9 different concentrations.

Fig. 15

ICW analysis visualizing the plates. Plates 1–3 in green are for visualization of the primary antibody, in red for visualization of the cell stain depicting cell number, and the merged plate. Compounds are plated in duplicate with the highest concentration on the left. BG is an empty well for background reading.

Fig. 16

ICW Analysis highlighting 2 PROTACs and the CDK4/6 inhibitor, palbociclib. (A) YX-02-107 and AC-03-092 (BSJ-03-123) are reported CDK6 degraders and Palbociclib is a clinically approved CDK4/6 inhibitor. In this cell line and under these analysis conditions YX-02-107 (DC50=0.01μM) and AC-03-092 (DC50=0.06μM) show potent CDK6 degradation consistent with their reported values. Palbociclib shows a modest increase in CDK6 levels after a 24-h treatment. (B) Compound treatment did not reduce cell number. (C) Structures of the compounds are shown.

Fig. 17

Western blot analysis of CDK6 protein levels in SK-MEL-2 cells after 24h treatment. SK-MEL-2 cells were treated with YX-02-107 at six different concentrations and Palbociclib at three different concentrations for 24h prior to Western blot analysis. CDK6 has a molecular weight of 36kDa, β-Actin (molecular weight=42kDa) was chosen as a loading control, expression levels of this protein do not vary drastically due to cellular treatment. Protein loading=15μg.

Fig. 18

The plate map for seeding SK-MEL-2 cells. Columns 1, 2, 11 and 12 are not used for analysis due to edge effects and are filled with PBS to help avoid evaporation.

Fig. 19

The plate map for the primary and secondary antibody dilution. The out-lying wells are not used for the assay.

Fig. 20

Signal Linearity. (A) Signal linearity normalized to CellTag 700. (B) Signal linearity for CellTag700 at various cell densities. (C) Signal to Noise ratio calculated for various primary antibody dilutions and cell number.

Fig. 21

(A) Plate Image for visualization of PARP1 with secondary antibody IRDye 800CW. (B) Plate Image for visualization with CellTag 700. (C) Plate Image A and B merged.

Fig. 22

Western Blot with PARP1 in SNU-719.

Fig. 23

ICW Analysis for 3 compounds in SNU719 cells. (A) AC-030-086 and AC-03-088 are PARP1 degraders and JG-01-070 is a control compound. In this cell line under these analysis conditions AC-03-086 (DC50=42nM) and AC-03-088 (DC50=65nM) show potent PARP1 degradation. The control compound JG-01-070 did not decrease PARP1 levels. (B) AC-03-088 treatment reduced cell number at concentrations around 1–3μM.

Fig. 24

ICW analysis visualizing the plates. Plate in green is for visualization of the primary antibody, in red for visualization of the cell stain depicting cell number, and the merged plate. Compounds are plated in duplicate with the highest concentration on the left. BG is an empty well for background reading.