Irdye 800cw goat anti mouse immunoglobulin g igg

DMEM, DMEM GlutaMax, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Invitrogen/ThermoFisher Scientific. Easy-Tag [³⁵S]-Cys/Met was purchased from PerkinElmer Life Sciences. S-protein-agarose beads and S-tag antibody were purchased from EMD Millipore. Endo H, PNGaseF, the Protoscript II first strand cDNA synthesis kit, and all cloning reagents were purchased from New England Biolabs. FastStart SYBR Green qPCR mix was purchased from Roche Diagnostics, and all primers were acquired from IDT DNA. IRDye 800CW goat anti-mouse immunoglobulin G (IgG) and IRDye 680RD goat anti-rabbit IgG were purchased from Li-COR Biosciences. Protein A sepharose CL-4B was purchased from GE Healthcare. Antibodies directed toward the following antigens were also purchased: calnexin (Enzo Life Sciences), CRT (ThermoFisher Scientific), E-cadherin (GeneTex), pan-cadherin (Sigma), ERp57 (a gift from Taku Tamura, Akita University, Japan), GM130 (BD Biosciences), and KDEL (Enzo Life Sciences). TMTC3 and TMTC4 cDNA were created by isolating RNA (HEK293T cells) and cloned into pcDNA3.1 A−, a plasmid harboring a C-terminal S-tag, using standard molecular biology techniques. All other chemicals were obtained from Sigma.

Cells were lysed in NP-40 lysis buffer (Table S5), incubated on ice for 20 min, and then centrifuged at 15,000 × g for 20 min. Protein concentration was determined by Pierce BCA Protein Assay Kit, and 20 μg protein was denatured with 2.5 μL LDS sample buffer and 1 μL reducing agent (NuPAGE) at 37°C for 30 min. Samples were resolved on NuPAGE 4%–12% BisTris protein gels with mass standards of 10–250 kDa (LI-COR). Proteins were transferred to Immobilon-FL polyvinylidene fluoride (PVDF) membranes (Millipore). Membranes were blocked in Odyssey Blocking Buffer (LI-COR Biosciences), immunostained with anti-CFTR or anti-α-tubulin followed by IRDye 800CW goat anti-mouse immunoglobulin G (IgG; LI-COR Biosciences), visualized, and quantified on an Odyssey IR Imager (LI-COR Biosciences) (Tables S3 and S4).