Hrp conjugated goat anti rabbit and hrp conjugated goat anti mouse

Worm lysates were loaded on a 4–20% Mini-Protean TGX Precast Gel (Bio-Rad). The gel was run at 120V for 15 minutes to fully collapse samples and then 180V. Transfer to PDVF (Thermo Scientific) was performed at 4°C at 350mA for 1.25 hr. Membranes were blocked in 5% non-fat milk/TBST for one hour at room temperature and incubated overnight at 4°C with the following primary antibodies diluted in blocking reagent: 1 μg/mL mouse α-alpha-tubulin (DM1A, EMD Millipore), 1 μg/mL rabbit α-LEM-2 (Novus Biologicals), 1 μg/mL rabbit α-CHMP-7 (Shankar et al., 2022 (link)) and rabbit α-BAF-1 (Gorjanacz et al., 2007 (link)). The following day, membranes were briefly rinsed in TBS followed by three five-minute washes in TBST. Membranes were then incubated with appropriate secondary antibodies for 1.25 hrs at room temperature. Secondary antibodies were diluted 1:10,000 for horseradish peroxidase (HRP)-conjugated goat-anti-rabbit and HRP-conjugated goat-anti-mouse (Thermo Fischer Scientific). Membranes were again briefly rinsed in TBS followed by three five-minute washes in TBST. Membranes were incubated with Clarity Max Western ECL Substrate (BIO-RAD) for five minutes before imaging (BioRad ChemiDoc MP Imaging Systems).

Cells were cultured in the given concentration of drug/s for 4h, and then lysed in 10mM Tris/HCl buffer (pH 7.4) containing 137mM NaCl, 10% Glycerol, 1% NP40, 10mM β-glycerophosphate, 2mM sodium vanadate, 2mM NaF, 10mM sodium pyrophosphate, and cOmplete^TM EDTA-free protease inhibitor (Sigma-Aldrich). Lysates were subjected to SDS-PAGE with pre-cast 4-20% TGX gels (ThermoFisher), proteins transferred to nitrocellulose membranes, which were blocked for at least 1h at room temperature in either 5% skim milk powder in 50mM Tris/HCl buffer (pH 7.4) containing 150mM NaCl and 1% Tween 20 if imaging on a LAS-4000 (GE Healthcare), or Li-Cor PBS Odyssey Blocking Buffer if imaging on an Odyssey (Li-Cor, Lincoln, NE). Primary antibodies used were ATF4, XBP1, PSMB5, PSMB6, PSMB7 (Cell Signaling Technology), and β-actin (Sigma-Aldrich). Secondary antibodies used were HRP-conjugated goat-anti-rabbit and HRP-conjugated goat-anti-mouse (ThermoFisher), and goat-anti-rabbit IR and goat-anti-mouse IR 680RD (Li-Cor).