Acc 020

Western blotting was performed as previously described 18 (link). Briefly, equal amounts of total protein per sample were separated on a polyacrylamide gel containing 0.1% SDS and then transferred to PVDF membrane. The membranes were incubated overnight with primary antibodies of TRPC5 (1:1000, ACC-020, Alomone Labs), β-actin (1:1000, #4967, Cell Signaling Technology), TSG101 (1:1000, ab125011, Abcam), CD81 (1:1000, ab109201, Abcam), fibroblast activation protein (FAP) (1:1000, ab53066, Abcam), a-SMA (1:1000, ab32575, Abcam), FSP-1 (1:1000, ab124805, Abcam) and Vimentin (1:1000, ab193555, Abcam) at 4°C, followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:500, ab205718, Abcam) for 1 h at room temperature. The protein bands were developed with an enhanced chemiluminescence kit (E411-04, Vazyme), and observed using ChemiDocXRS+ gel imager (Bio-Rad).

Whole-cell protein was obtained using RIPA containing 1 mM PMSF. An equal quantity of total proteins was electrophoresed on 8% polyacrylamide gel containing 0.1% SDS and then transferred to PVDF membrane. After blocked with phosphate-buffered saline tween containing 5% non-fat milk, the PVDF membranes were incubated with the primary antibodies anti-TRPC5 (ACC-020, Alomone labs, Jerusalem, State of Israel) (1:500), anti-caspase-3 (ab32351, Abcam Biotechnology, Cambridge, MA, USA) (1:500), anti-glucose transporter 1 (GLUT1) (ab115730, Abcam Biotechnology) (1:1000), β-actin (AA128, Beyotime Biotechnology) (1:1000) and subsequently with the corresponding secondary antibodies [goat anti-rabbit IgG (A0208, Beyotime Biotechnology) and goat anti-mouse IgG (A0216, Beyotime Biotechnology)]. The bands were quantified using ImageJ software (NIH, Bethesda, MD). β-actin was used as the internal control for normalization.