Anti bid

Polyclonal antibodies against TMEM106A were prepared by immunizing rabbits with chemically synthesized TMEM106A peptides (Fig. S1A, rectangle sequences), purified by peptide affinity chromatography via CNBr-activated Sepharose™ 4 Fast Flow (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), according to the manufacturer's instructions. Other antibodies used in this study were: anti-β-actin/ACTB and anti-MYC (Sungene Biotech Tianjin, China), anti-PARP (Cell Signaling Technology, Beverly, MA, USA), anti-Bid (Santa Cruz, CA, USA), anti-tBid (ab10640; Abcam, Cambridge, UK) and DyLight 800/DyLight 680-conjugated secondary antibodies against mouse/rabbit IgG (Rockland, ME, USA). z-VAD-fmk (Promega, Madison, WI, USA), Hoechst 33342 and 5-aza-2′-deoxycytidine (5-Aza) (Sigma-Aldrich, St Louis, MI, USA), Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) were also used.

Cells were seeded in six-well plates (2.5 × 10⁵/well for 3-h treatment; 2 × 10⁵/well for 24-h treatment), allowed to adhere for 24 h and treated with TRAIL or APG350 for either 3 or 24 h. Whole-cell lysates were prepared using RIPA buffer and analyzed by western blot as described^{23 (link)}. Primary and secondary antibodies used were purchased from: Cell Signaling Technology, Frankfurt/ Main, Germany (anti-caspase-8, anti-PARP, anti-phospho-p38, anti-phospho-IκBα, anti-phospho-p42/44, anti-phospho-JNK, anti-p38, anti-p42/44, anti-JNK, anti-mouse-IgG-HRP, anti-rabbit-IgG-HRP), BD Bioscience, Heidelberg, Germany (anti-Bcl-xL), R&D Systems, Minneapolis, Canada (anti-Bid), Santa Cruz, Heidelberg, Germany (anti-IκBa, anti-goat-IgG-HRP) and Sigma-Aldrich (anti-β-actin).

The Ni²⁺-NTA agarose were obtained from QIAGEN (Dusseldorf, Germany) and Protein A+G Agarose beads were purchased from Beyotime Biotechnology (Shanghai, China). N-ethylmaleimide (NEM) were purchased from Sigma-Aldrich (St. Louis, MO). The complete protease inhibitor cocktail, the PhosSTOP phosphatase inhibitor cocktail and the In Situ Cell Death Detection Kit was purchased from Roche (Basel, Switzerland). Primary antibodies used in this research were listed below: anti-annexin A1 (sc-12740, 1:1000), anti-HA (sc-7392, 1:1000), anti-Flag (sc-166355, 1:1000), anti-α-tubulin (sc-100585, 1:2000), anti-β-actin (sc-47778, 1:2000), anti-PKC (sc-17769, 1:1000), anti-Bid (sc-11423, 1:1000) were purchased from Santa Cruz Biotechnology (Dallas, TX); anti-SUMO2/3 (#4971, 1:1000), anti-Myc (#2276, 1:1000), anti-Histone H3 (#4499, 1:2000), anti-cleaved caspase-3 (9664, 1:1000), anti-cleaved PARP (5625, 1:1000), anti-cleaved caspase-9 (#20750, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA); anti-SENP6 (HPA024376, 1:1000), anti-His (SAB1306085, 1:1000) were purchased from Sigma-Aldrich, and anti-TRPM7 (ab232455, 1:500) were purchased from Abcam (Cambridge, UK). All other general reagents were purchased from commercial suppliers and used as received.