Mouse monoclonal anti flag f3165

Primary antibodies used for immunoblotting were as follows: mouse monoclonal anti-T7 (69522, Merck, 1 μg/ml), mouse monoclonal anti-Flag (F3165, Sigma-Aldrich, 1:1000), mouse monoclonal anti-Myc IgG-9E10 (CRL-1729, ATCC, 1 μg/ml), mouse monoclonal anti-ubiquitin IgG-P4D1(sc-8017, Santa Cruz Biotechnology, 1:1000), mouse monoclonal anti-hamster HMGCR IgG-A9 (CRL-1811, ATCC, 2 μg/ml), mouse monoclonal anti-clathrin heavy chain (610500, BD Biosciences, 1:1000), mouse monoclonal anti-Actin (A3853, Sigma, 1:5000), polyclonal anti-HMGCR antibody (H2) was raised against a C-terminal sequence (aa410-aa888) of human HMGCR in our laboratory [43 (link)]. Horseradish peroxidase-conjugated donkey anti-mouse (715-005-151, 1:5000) and anti-rabbit (711-005-152, 1:5000) secondary antibodies were obtained from Jackson ImmunoResearch Laboratories.
Primary antibodies used for immunofluorescence staining were as follows: rabbit polyclonal anti-GM130 (G7295, Sigma, 1:300), rabbit polyclonal to calnexin (ab22595, Abcam, 1:300). Alexa Fluor 488-labeled donkey anti-mouse IgG (A-21202, 1:500) and Alexa Fluor 555-labeled donkey anti-rabbit IgG (A-31572, 1:500) secondary antibodies were obtained from Invitrogen.

HEK293 transfected with single pTAL10-MSTN or co-transfection with MSTN-L and MSTN-R were lysed 48–72 hours post transfection with cold RIPA buffer supplemented with protease inhibitors (1 mM PMSF and 1 mM Benzamidine). Protein concentration was determined by using Bradford Protein Assay Kit (IBI Scientific, Peosta, IA). 20 μg of protein were resolved on a 4-20% Mini-PROTEAN TGX Precast Gel (Bio-Rad Laboratories, Hercules, CA) and transferred onto a PVDF membrane (Millipore, Billerica, MA). Immunoblotting was done with the following antibodies: mouse monoclonal anti-FLAG (F3165; Sigma-Aldrich, Saint Louis, MO), polyclonal anti-HA-Tag antibody (Clontech, Mountain View, CA), mouse monoclonal anti-GAPDH (Millipore, Billerica, MA), and HRP conjugated goat anti-mouse IgG (Millipore, Billerica, MA). Membranes were developed using ECL 2 Western Blotting Substrate (Pierce Biotechnology, Rockford, IL) and images were captured using ChemiDoc XRS + system (Bio-Rad Laboratories, Hercules, CA).