Mouse anti myc

Cells were cotransfected with his-tagged hTRPM8pcDNA4 plasmid and HA-tagged TCAF1 or myc-tagged TCAF2, and were washed two times, fixed with 4% formaldehyde–1× PBS for 15 min, washed three times, then permeabilized in PBS-gelatin (1.2%) complemented with 0.01% Tween 20 and 100 mM glycine for 30 min at 37°C. Afterward, cells were incubated with primary antibodies: 1:200 goat polyclonal anti-TRPM8 antibody (Antibodies Online), rabbit anti-HA (1:100; Abcam), and mouse anti-myc (1:100; Invitrogen) in PBS-gelatin at 37°C for 1.5 h. After thorough washes, the slides were treated with the corresponding secondary antibodies: donkey Rhodamine Red-X–labeled anti–goat (dilution 1:300; Jackson ImmunoResearch Laboratories, Inc.) and donkey Alexa Fluor 488–labeled anti–rabbit (dilution 1/250; Jackson ImmunoResearch Laboratories, Inc.) diluted in PBS-gelatin for 1 h at room temperature. The slides were then incubated with 0.3% Sudan Black in 70% ethanol in order to reduce autofluorescence, washed two times, and mounted with Mowiol. Fluorescence analysis was performed using a confocal microscope (LSM 700; Carl Zeiss, Inc.) and ImageJ analysis software.

Cells were cotransfected with a his-tagged hTRPM8pcDNA4 plasmid and human influenza agglutinin (HA)-tagged TCAF1 or myc-tagged TCAF2, washed twice with PBS, and incubated for 60 min on ice in lysis buffer (1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, 10 mM NaKPO₄, pH 7.2, and anti-protease cocktail; Sigma-Aldrich). After centrifugation (12,000 g for 10 min at 4°C) of the lysates, protein concentration was determined by the BCA assay (Thermo Fisher Scientific), and equal amount of supernatants were incubated overnight at 4°C with mouse anti-his antibody (Invitrogen) immobilized on protein A/G PLUS agarose beads (Santa Cruz Biotechnology, Inc.). The pellet was washed three times, resuspended in SDS sample buffer, and heated at 37°C for 30 min, separated on 10% wt/vol SDS-PAGE gels, and analyzed by immunoblotting using rabbit anti-HA (1:2,000; Abcam), mouse anti-myc (1:500; Invitrogen), mouse anti-actin (1:5,000; Sigma-Aldrich), mouse anti-calnexin (1:2,000; EMD Millipore), rabbit anti-TRPM8 (1:1,500; Alomone Labs Ltd), and rabbit anti-TRPV6 (1:200; Santa-Cruz Biotechnology, Inc.) antibodies.

HEK293T cells were seeded in 12‐wells plates the day before transfection. One microgram of vectors (pcDNA3.1) encoding mutant soluble Myc‐tagged EDA1 protein was transfected into each well using Lipofectamine 2000 (Invitrogen), and vectors encoding wild‐type soluble Myc‐tagged EDA1 and vector pcDNA3.1 were used as positive and negative controls, respectively. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 µg/ml streptomycin. Forty‐eight hours after the transfection, the cells and supernatants were harvested separately for western blot study, and the Myc‐tagged EDA1 proteins in cell lysates and supernatants were detected by western blots as described previously (Liu et al., 2019 (link)). For immunofluorescence studies, the cells were fixed with 4% paraformaldehyde for 20 min, then permeabilized before blocking in PBS with 5% horse serum for 1 hr and then stained overnight with mouse‐anti‐Myc (Thermo Fisher Scientific). Goat anti‐mouse 488 (Thermo Fisher Scientific) was used as a secondary antibody and DAPI (Thermo Fisher Scientific) was used for counterstaining. Images were captured using a microscope (Olympus IX83).