Criterion tgx precast sds page gels

Trypsinized cells were pelleted and washed with ice-cold phosphate-buffered saline (PBS). Cells were lysed in cell lysis buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM Na₄P₂O₇, 1 mM β-glycerol phosphate, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 10% glycerol, 2 mM activated Na₃VO₄, 1 mM NaF and freshly added 1x protease inhibitor cocktail), and centrifuged at 16,000x g at 4 °C for 10 min. Proteins were then resolved on 4-15% Criterion TGX precast SDS-PAGE gels (Bio-Rad) and electro-transferred to PVDF membranes (Bio-Rad Trans-Blot Turbo transfer system). Primary antibodies (Abs) were anti-FLAG M2 (Sigma Aldrich F1804), anti-XBP1s (Clone 143F, BioLegend) and anti-TBP (TATA-box binding protein) Abs. TBP was used as a loading control. Blots were imaged and quantified using ImageJ.

Cells were trypsinized, pelleted and washed with cold phosphate-buffered saline (PBS). Nuclei were isolated as described in CUT&RUN below, lysed in cell lysis buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM Na₄P₂O₇, 1 mM β-glycerol phosphate, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 10% glycerol, 2 mM activated Na₃VO₄, 1 mM NaF and fresh added 1× protease inhibitor cocktail), and centrifuged at 16 000 × g at 4°C for 10 min to remove nuclear debris. Proteins were resolved on 4–15% Criterion TGX precast SDS-PAGE gels (Bio-Rad) and transferred to PVDF membranes with Bio-Rad Trans-Blot Turbo transfer system. Primary antibodies used were anti-FLAG M2 (Sigma Aldrich F1804), anti-XBP1s (Clone 143F, BioLegend) and anti-TBP (TATA-box binding protein) as a loading control. Blots were imaged and quantified using ImageJ.