Fusion fx chemi imager

Cells were lysed in RIPA buffer (150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 25 mM Tris pH 7.4) containing freshly added protease inhibitors (Halt Protease Cocktail, Thermo Fisher, Waltham, MA, USA). Samples were incubated on ice for 10 min, sonicated 4 cycles of 30 s on/off (Biorupter, Diagenode, Denville, NJ, USA) and centrifuged (21,100 ×g, 10 min, 4 °C). The DC protein assay (BioRad, Hercules, CA, USA) was used to quantify protein concentrations. Then, 30 μg of protein was separated using SDS-PAGE and immunoblotting performed^{65 (link)}. Primary antibodies used were rabbit anti-TMPRSS2 (1:100, ab92323, Abcam, Cambridge, UK), mouse anti β-actin (1:8000, ab8226, Abcam), rabbit anti-AR (1:2000, ab74272, Abcam) and mouse α-tubulin (1:10,000, B-5-1-2, Sigma Aldrich). Secondary HRP-conjugated antibodies (Sigma Aldrich) were used at 1:2000. Proteins were visualised using Immobilon Forte HRP substrate (Merck Millipore, MA, USA) and a Fusion FX Chemi Imager (Vilber Lourmat, Collégien, France).

Samples were lysed in RIPA buffer (150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 25 mM Tris pH 7.4) containing freshly added protease inhibitors (Halt Protease Cocktail, Thermo Fisher, Waltham, MA, USA) and prepared as previously described [21 (link)]. The DC protein assay (BioRad, Hercules, CA, USA) was used to quantify protein concentrations. 60 μg of protein was separated using SDS-PAGE and immunoblotting performed. Primary antibodies used were rabbit anti-ERα (1:1000, sc-543, Santa Cruz, TX, USA), mouse α-tubulin (1:10,000, B-5-1-2, Sigma-Aldrich). Secondary HRP-conjugated antibodies (Sigma-Aldrich) were used at 1:2000. Proteins were visualised using Immobilon Forte HRP substrate (Merck Millipore, MA, USA) and a Fusion FX Chemi Imager (Vilber Lourmat, Collégien, France).