Anti ha beads

Plat-E cells were transfected with pMX-Cecr2-HA/pMX-Cecr2 ΔDDT-HA and pMX-Smarca1-3×FLAG at the same time. Thirty-six hours after transfection, cells were digested, counted, and lysed. One milliliter lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 2 mM EDTA, 1% NP-40, and protease inhibitors) was used to lyse 1 × 10⁷ cells. Cells were lysed for 30 min at 4 °C. The lysates were centrifuged (13,000g for 10 min) and only the supernatant was collected. Immunoprecipitation was performed by 400 μl supernatant and 20 μl anti-HA beads (88837, Thermo Scientific) for 40 min at room temperature. Beads were washed with lysis buffer for five times and then boiled in SDS loading buffer for 10 min to resuspend sample. Antibodies used for coIP were anti-HA (3724s, CST); anti-FLAG (F1804, Sigma).

Either HA-tagged Cdh1 and Myc-tagged Chk1 mutant or HA-tagged Cdh1 (or mutants) and Flag- βTRCP1were expressed where indicated in 293T cells for 30 h. Cells were treated with MG-132 (10 µM for 5 h) prior to lysis. Cell extracts were generated in EBC buffer, 50 mM Tris (pH 8.0), 120 mM NaCl, 0.5% NP40, 1 mM DTT, and protease and phosphatase inhibitors tablets (Thermo Fisher Scientific).For immunoprecipitation, equal amounts of cell lysates were incubated with the indicated antibodies conjugated to protein G beads (Invitrogen) or anti-HA beads (15 µl per IP, Thermo Scientific) respectively from 4 h to overnight at 4 °C. The beads were then washed with EBC buffer including inhibitors. Binding to immobilized GST proteins was performed as described previously^{33 (link)}. Immunoprecipitation samples or equal amount of whole-cell lysates were resolved by SDS-PAGE, transferred to PVDF membranes (Milipore) probed with the indicated antibodies, and visualized with the LiCor Odyssey infrared imaging system.

Cells were collected, washed, lysed with NP-40 lysis buffer (Beyotime, Cat# P0013F) and incubated on ice for 30 min. Lysates were centrifuged at 4 °C for 15 min at 12,000 rpm, and the supernatants were collected. A Bradford (Beyotime, Cat# P0006C) kit was used to determine protein concentrations. For IP experiments, samples containing one milligram of protein were incubated with the indicated anti-Flag-M2 beads (Sigma, Cat# M8823) or anti-HA beads (ThermoFisher Scientific, Cat# 88838) for 1 h at room temperature. After incubation, the beads were washed with lysis buffer five times. After washing, the beads were mixed with LDS Sample Buffer (Invitrogen, Cat# B0007) and boiled for 10 min for western blot analysis.

Membrane fractions were resuspended and incubated in MELB supplemented with 0.5 mM PEG-maleimide (5 kDa, Sigma-Aldrich) and cOmplete protease inhibitors (Roche) at room temperature for 30 min. The reaction was then quenched by addition of n-ethylmaleimide at a final concentration of 20 mM. Proteins were solubilised in 1% w/v digitonin for 30 min at 4°C prior to immunoprecipitation of HA-VDAC2 with anti-HA beads (Thermo Fisher) for 1 h at 4°C. Proteins were eluted from the beads by boiling for 5 min in SDS-PAGE sample buffer prior to analysis on SDS-PAGE.

Cells were lysed in IP lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, supplemented with protease and phosphatase inhibitors) and incubated on a rotator at 4 °C for 10 min. Chromatin was then pelleted by centrifugation for 5 min at 1000×g and then digested with 100 U/mL Benzonase at room temperature in Benzonase buffer (2 mM MgCl₂, 50 mM Tris, pH 7.4, 150 mM NaCl). For denaturing IPs, samples were supplemented with SDS (final concentration 1%) and incubated on ice for 10 min. Denaturing IPs were quenched with Triton X-100 (to a final concentration of 1%). Lysates were supplemented with ethidium bromide (50 μg/mL) and incubated with anti-FLAG M2 agarose (Sigma-Aldrich), Strep-Tactin Sepharose (IBA), or GFP-Trap agarose (Chromotek) for 1–3 h(s) on a rotator at 4 °C, washed three times with IP wash buffer (150 mM NaCl, 50 mM Tris–HCl; 0.5 mM EDTA), and resuspended in 2 × Laemmli buffer. For TAP experiments, cells were grown on 245 × 245 mm cell culture dishes (two per condition). Immunoprecipitation of TEX264-FLAG was performed as described above. After TEX264-FLAG was captured, samples were boiled for 5 min in 1% SDS then quenched with 1% triton and incubated with anti-HA beads (Thermo Fisher) for 16 h at 4 °C with rotation. The next day samples were washed three times in IP wash buffer and eluted in 2× Laemmlli buffer.