Alexa 594 anti calnexin antibody af18

Cells were fixed with 2% paraformaldehyde (PFA) w/o methanol (15 min, Thermo Fisher Scientific, Darmstadt, Germany), permeabilized, and blocked with 0.5% Triton X-100 (Thermo Fisher Scientific, Waltham, USA) in 2% BSA (PAN-Biotech, Aidenbach, Germany) for 60 min. ER stress markers included: Alexa 647 anti-ATF-4 antibody (B-3, 1:50, Santa Cruz), Alexa 594 anti-calnexin antibody (AF18, 1:50, Santa Cruz), and Alexa 488 anti-cytochrome c (1:50, Biolegend). For stemness, antibody mixtures, either containing anti-GFAP (1:200, Alexa Fluor 594, BioLegend, San Diego, USA) and anti-A2B5 (1:200, Alexa Flour 647, BioLegend, San Diego, USA), or anti-Oct-4 (1:500, Alexa Fluor 647, BioLegend, San Diego, USA) and anti-Nanog (1:500, Alexa Flour 488, BioLegend, San Diego, USA) were added and staining was done at 4 °C overnight. The next day, GFAP/A2B5-antibody mix was stained with Phalloidin green (1:50, BioLegend, San Diego, USA). Nuclei were counterstained with DAPI (1:1.000, Biomol, Hamburg, Germany) and cells were analyzed using a Zeiss microscope Axio Observer 7 (Zeiss, Oberkochen, Germany).

GBM cells were treated for 2 x 72 h and further treated as described [16 (link)]. Additionally, CDKN2A/p16INK4a antibody (JC8) (1:50, Santa Cruz), Alexa Fluor® 488 p21 Waf1/Cip1 (1:300, Cell Signaling), Alexa Flour® 594 anti-p53 antibody (1:50, Biolegend), Alexa Fluor® 594 anti-H2A.X Phospho (Ser139) antibody (1:100, Biolegend), FITC anti-GADD45 antibody (1:100, Biorbyt), PE anti-β-Catenin antibody (1:75, Biolegend), anti-AXIN2 antibody (1:50; Thermo Scientific), Alexa 647 anti-ATF-4 antibody (B-3, 1:50, Santa Cruz), Alexa 594 anti-calnexin antibody (AF18, 1:50, Santa Cruz), and Alexa 488 anti-cytochrome c (1:50, Biolegend). For AXIN2, a secondary DyLight 488-conjugated anti-rabbit antibody was applied (1:100; Biolegend). Nuclei were counterstained with DAPI. Cells were analyzed on a Zeiss Elyra 7 Confocal Laser Microscope. Protein extraction and western blotting was done as described [11 (link)]—a list of antibodies is given in STable 1. All data sets were normalized to GAPDH, which was used as housekeeping control (please see SFig. 6 for original western blots).