Ta50011

Cells kept on ice were lysed with 1x RIPA lysis buffer (Millipore) supplemented with protease inhibitors (Complete, Roche). Membranes were blocked in 5% fat-free milk 1 hr at RT, incubated O/N at 4 °C with primary antibody, and then for 2 hr at RT with a secondary antibody conjugated to horseradish peroxidase (HRP). Chemiluminescence detection used ECL-plus reagent (Pierce). To ensure equal protein loading across gels, membranes were stripped (Restore western blot stripping buffer; Pierce) and re-probed with a loading control antibody. For dot blots, protein samples were spotted onto nitrocellulose membranes using a slot blot apparatus and immunoblotting was performed as described above. Antibodies for: MICU1 (ab102830, abcam; 1:200) and (HPA037480, Sigma; 1:300); MICU2 (ab101465, abcam 1:500) and (HPA045511; Sigma); MCU (HPA016480, Sigma; 1:500); EMRE (sc-86337; Santacruz) and (HPA032117, Sigma; 1:200); Tom20 (ab56783; abcam); Tim23 (611223; BD Biosciences); Tim44 (ab168649; abcam); Oxa1L (66128-1-Ig; proteintech); HSP60 (ab13532; abcam); anti-V5 (R960-25, Life technologies; 1:1000); anti-actin (A2228, Sigma 1:2000); anti-flag (TA 50011, Origene 1:500); anti-mouse IgG-HRP (7076S, Cell Signaling; 1:1000) and anti-rabbit IgG-HRP (7074S, Cell Signaling; 1:1000) were used in the study. All experiments were done in triplicate.

Proteins were extracted using RIPA buffer system. Protein samples were run on NuPage 4 to 12% bis-tris gel (Invitrogen), transferred to nitrocellulose membranes (Bio-Rad Laboratories), and blocked with 3% bovine serum albumin (BSA) in tris buffer saline–Tween 20 (TBST). Membranes were incubated with anti-CHRM4 rabbit polyclonal antibody (1:2000; TA321192, OriGene), anti-GAPDH mouse monoclonal antibody (1:10000), anti-ZFP36L2 antibody (1:1000; ab70775, Abcam), anti-CREB antibody (1:8000; 17–600, EMD Millipore), anti-GATA2 antibody (1:1000; ab22849, Abcam), anti-KIT antibody (1:1000; ab46758, Abcam), or anti-DDK antibody (1:2000; TA50011, OriGene) in 3% BSA overnight at 4°C. Membranes were washed six times with TBST for 5 min. Secondary antibodies against rabbit IgG (1:10000) and mouse IgG (1:10000) were incubated with membranes for 2 hours at room temperature in 3% BSA solution. Membranes were washed again six times with TBST for 5 min. Western Lightning Plus Enhanced Chemiluminescence substrate (PerkinElmer) was added to membranes for 45 s, and autoradiographs were developed. C-terminal DDK-tagged CHRM4 cell lysates were from OriGene (LY400245). HEK293T cells were transfected with plasmid encoding DDK-tagged CHRM4 or control vector, followed by 48 hours of culture, and cells were lysed in modified RIPA buffer.

Western blotting was conducted as previously described (Costantini et al. 2007 (link), Jonas et al. 2010 (link), Peng et al. 2014 (link)). The following primary antibodies were used in this study: anti-CREB (Cell Signal 4820; RRID:AB_1903940), anti-c-FOS (Abcam #ab134122), anti-c-JUN (Cell Signal 9165; RRID:AB_2130165), anti-p53 (Cell Signal #9282S; RRID:AB_331476), anti-REST (Millipore #17–10456; 2017), anti-acetylated lysine (Abcam #ab409; RRID:AB_308707), anti-DDK (Origene TA50011; RRID:AB_2622345), and anti-Myc (Santa Cruz Biotechnology sc-40; RRID:AB_627268). Donkey anti-rabbit Alexa Fluor 800-conjugated secondary antibodies (LI-COR Biosciences Cat# 925–32213; RRID:AB_2715510 and LI-COR Biosciences Cat# 925–32210; RRID:AB_2687825) were used for infrared imaging (LICOR Odyssey Infrared Imaging System; LI-COR Biosciences).

Cells transfected with DDK-tagged human C6 were lysed in SDP buffer as described above. 200 μg total protein were subjected to immunoprecipitation over night at 4 °C using 2.5 μl anti-DDK antibody (Origene TA50011) coupled to 20 μl magnetic protein G beads (Dynal, Invitrogen). Immunoprecipitates and cell lysates were subjected to SDS-PAGE and Western blot as described above.