Image lab 6

Cells were lysed, and protein levels were determined by SDS-PAGE coupled to Western blotting. Additional details for this protocol are provided in the Supplementary Materials. Antibodies were obtained commercially and included GATA4 (1:1000, Cell Signaling Technology, Danvers, MA, USA) and COL1α1 (1:1000, Aviva Systems Biology, San Diego, CA, USA). The secondary antibody was anti-rabbit (1:10,000, Santa Cruz Biotechnology Inc., Dallas, TX, USA). Image analysis was performed using Image Lab 6.0 software from Bio-Rad (Berkeley, CA, USA).

Total protein was extracted from four LA-derived primary cell lines. Protein was separated by SDS-PAGE and transferred to a PVDF membrane as previously described [43 (link)]. The iBind Flex (cat# SLF2000, ThermoFisher Scientific, Waltham, MA, USA) was used for antibody binding with the primary antibodies for OCT4 (1:1000; cat# ab109183, Abcam, Cambridge, UK), NANOG (1:1000; cat# ab109250, Abcam, Cambridge, UK), SOX2 (1:500; cat# 48-1400, ThermoFisher Scientific, Waltham, MA, USA), KLF4 (1:000; cat# NBP2-24749, Novus Biologicus, Centennial, CO, USA), c-MYC (1:1000; cat# ab32072, Abcam, Cambridge, UK) and α-tubulin (1:2000; cat# 62204, ThermoFisher Scientific, Waltham, MA, USA). Secondary antibodies used included goat anti-rabbit HRP (1:1000; cat# ab6721, ThermoFisher Scientific, Waltham, MA, USA) and goat anti-mouse alexa488 (1:1000; cat# A21202, ThermoFisher Scientific, Waltham, MA, USA). Clarity Western ECL (cat# 1705061, Bio-Rad, Los Altos, CA, USA) was used for visualizing HRP detected protein bands and the ChemiDoc MP Imaging System (Bio-Rad Laboratories, Los Altos, CA, USA) and Image Lab 6.0 software (Bio-Rad Laboratories, Los Altos, CA, USA) were used for band detection and analysis. NTERA-2 cells were used as a positive control, and α-tubulin was used as a loading control.

Fresh frozen tissue lysis and Western blotting (WB) were performed as previously described [14 (link)]. The primary antibodies are provided in Additional file 1: Table S1, and many were previously tested in autopsy tissue [14 (link)]. WBs for each antibody were repeated at least twice, with similar results. The densitometric analysis was performed by scanning the X-ray films with optimal exposures on a ChemiDoc™ Touch imager (Bio-Rad, Hercules, CA). The bands were quantified by using Image Lab 6.0 software (Bio-Rad). Individual protein values were normalized to the corresponding actin or IDH1-R132H values, except for phosphoprotein values that were normalized to the corresponding unphosphorylated protein values. Minus values were manually adjusted as zero. Results were expressed as percent of the highest normalized values.