Mouse anti flag m2

For western immunoblotting, samples were fixed in TCA, acetone-washed and whole cell extracts prepared by bead-beating in TE containing protease inhibitors before running on SDS-PAGE and transferring to nitrocellulose membrane. Antibodies used were mouse anti-Ha (HA11, Covance) at 1:1000 dilution, mouse anti-GFP (Roche) at 1:1000, mouse anti-V5 (SV5-Pk1, Bio-Rad) at 1:2000, mouse anti-Flag (M2, Sigma) at 1:1000, rabbit anti-Pgk1 (lab stock) at 1:10000, rabbit anti-Kar2 (lab stock) at 1:20000, sheep anti-mouse-HRP (GE Healthcare) at 1:5000, donkey anti-rabbit-HRP (GE Healthcare) at 1:10000, donkey anti-mouse-IRDye 800CW (LI-COR Biosciences) at 1:10000 and donkey anti-rabbit-IRDye 680RD (LI-COR Biosciences) at 1:10000. Quantitative western blotting was performed using an Odyssey CLx Infrared Imaging System (LI-COR Biosciences) and quantified using ImageStudio 5.2.5 (LI-COR Biosciences).

Immunoblotting analyses were carried out as described previously^{24 (link)}. Digital images were captured using the LAS-1000 Plus (Fujifilm). The following primary antibodies were used: mouse anti-Flag M2 (Sigma), anti-Myc 9E10 (Santa Cruz), and anti-β-actin 2F3 (Wako); rabbit anti-Myc and anti-SP-B (Santa Cruz), anti-CtBP1, anti-CtBP2, anti-SP-C, anti-Muc1 and anti-Abca3 (Abcam), anti-Foxp1 (CST), anti-Foxp2 (Sigma), anti-phospho-ERK1/2 (CST), and anti-ERK2 (Santa Cruz). An affinity-purified anti-MCRIP1 antibody was made in-house as described previously^{24 (link)}. All antibodies were used at a dilution of 1:1000 for western blotting. Full size western blot images are shown in Supplementary Fig. 6.

All lysates were prepared using the buffer described under “Coimmunoprecipitation.” Lysates were mixed with 2× Laemmli buffer (Sigma-Aldrich) and incubated at 95°C for 10 min to denature proteins. Samples were loaded onto 4 to 20% mini-Protean TXG precast protein gels (Bio-Rad) and transferred onto 0.2-μm polyvinylidene difluoride (PVDF) membranes by semidry transfer (Bio-Rad). Membranes were then blocked in 5% milk for 1 h at room temperature, followed by incubation with appropriate primary antibodies for 1 h at room temperature. Primary antibodies used included rabbit anti-influenza A virus PB2 (GTX125926; GeneTex), mouse anti-FLAG M2 (F1804; Sigma-Aldrich), rabbit anti-Gaussia luciferase (E8023; New England BioLabs [NEB]), and rabbit anti-vinculin (EPR8185; Abcam). Following washing in TBS-1% Tween, membranes were incubated with secondary antibodies for 1 h at room temperature. Secondary antibodies included sheep anti-rabbit IgG–horseradish peroxidase (HRP) (AP510P; EMD Millipore) and goat anti-mouse IgG–HRP (STAR11P; Bio-Rad). Following washing, protein bands were detected by chemiluminescence using Amersham ECL prime Western blotting detection reagents (GE Healthcare). Protein bands were imaged using the Fusion Fx imaging system (Vilber Lourmat).

U2OS, 293TREx, and HepG2 cells were cultured in DMEM. K562 cells were cultured in RPMI. Culture media was supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin (ThermoFisher). U2-OS cells stably expressing GFP-HsATF6α were purchased from Thermo Scientific (084_01) and supplemented with 500 μg/ml G418 to maintain GFP-HsATF6α expression. HeLa CRISPRi cells expressing SFFV-dCas9-BFP-KRAB were previously described (Jost et al., 2017 (link)). Tunicamycin and thapsigargin were purchased from Sigma. Antibodies used were rabbit anti-GFP (ThermoFisher A11122), mouse anti-FLAG M2 (Sigma F1804), rat anti-GRP94 9G10 (abcam ab2791), rabbit anti-ACAA1 (Sigma HPA007244), rabbit anti-pmp70 (ab109448) for PFA fixation and (PA1-650) for methanol fixation, mouse anti-pmp70 (ab211533) for PFA fixation and (SAB4200181) for methanol fixation.

COS7 cells (monkey kidney fibroblast cell line, Korean Cell Line Bank #21651, Seoul, Korea) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco BRL) and 1% penicillin-streptomycin (Gibco BRL). Cells were transfected with an electroporation device (Neon Transfection System, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Parameters were set at 950 pulse voltage and 30 pulse width, and 2 pulses were used. Mouse anti-c-Myc (9E10), rabbit anti-c-Myc (A-14), rabbit anti-HA (Y-11), mouse anti-GST (B-14) and mouse anti-parafibromin (2H1) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Mouse anti-FLAG^® M2 and rabbit anti-FLAG^® antibodies were purchased from Sigma-Aldrich (Louis, MO, USA). Rabbit anti-CDC73 (D38E12), rabbit anti-K48 linkage-specific polyubiquitin (D9D5) antibodies were purchased from Cell Signal Technology (Danvers, MA, USA). Mouse anti-β-actin and rabbit anti-USP37 antibodies were purchased from Abcam (Cambridge, UK). Goat anti-Mouse IgG (Alexa Fluor 488) and goat anti-rabbit IgG (Alexa Fluor 594) antibodies were purchased from Invitrogen (Carlsbad, CA, USA).

The following primary antibodies were used for immunofluorescence labeling or immunoblotting: mouse anti-ACBD3 (Sigma) at a 1:1,000 dilution for immunofluorescence labeling and a 1:3,000 dilution for immunoblotting; mouse anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (Abcam) at a 1:1,000 dilution and mouse anti-Flag (M2; Sigma) at a 1:2,000 dilution for immunoblotting; rabbit antigiantin (Covance) at a 1:1,000 dilution, rabbit anti-TGN46 (LifeSpan Biosciences) at a 1:500 dilution, and goat anti-Salmonella antibody (CSA-1; Kirkegaard & Perry Laboratories) at a 1:200 dilution for immunofluorescence labeling; and mouse anti-HA (HA.11; Covance) at a 1:5,000 dilution for immunoblotting and a 1:1,000 dilution for immunofluorescence labeling.
Rhodamine Red X-conjugated donkey anti-mouse or anti-rabbit antibody, donkey anti-goat or donkey anti-rabbit–cyanine 2 (Cy2) antibody, and Cy5-conjugated donkey anti-mouse antibody were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA), for immunofluorescence labeling. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse antibodies were purchased from Dako for immunoblotting.