Anti α tubulin monoclonal antibody

Cells were lysed with radioimmunoprecipitation assay buffer and whole cell lysates were subjected to 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Thermo Fisher Scientific). The membranes were incubated with the anti-Sca-1 mouse monoclonal antibody (1:1,000), anti-c-kit rabbit polyclonal antibody (1:1,000; sc-168; Santa Cruz Biotechnology, Inc.), anti-osterix rabbit polyclonal antibody (1:1,000), anti-RUNX2 (runt-related transcription factor 2) rabbit polyclonal antibody (1:,000; sc-10758; Santa Cruz Biotechnology, Inc.), anti-fibronectin mouse monoclonal antibody (1:1,000; sc-59826; Santa Cruz Biotechnology, Inc.), anti-osteocalcin rabbit polyclonal antibody (1:1,000) and anti-osteopontin rabbit polyclonal antibody (1:1,000; sc-20788; Santa Cruz Biotechnology, Inc.). The membranes were then incubated with Novex^® alkaline-phosphatase-conjugated goat anti-rabbit polyclonal secondary antibody (WP20006; Thermo Fisher Scientific), or goat anti-mouse monoclonal secondary antibody (WP20007; Thermo Fisher Scientific) according to manufacturer’s instructions. The antibodies were detected using a chromogenic immunodetection system (WesternBreeze; Thermo Fisher Scientific) according to the manufacturer’s instructions. The anti-α-tubulin monoclonal antibody (Santa Cruz Biotechnology, Inc.) was used for the normalization of western blot analyses.

Monoclonal anti-α-tubulin antibody was purchased from Santa Cruz Biotechnology, monoclonal anti-AMPK, ERK, mTOR, ACC, p-AMPK, p-ERK, p-mTOR and p-ACC antibodies from Cell Signaling Technology (CST) and monoclonal anti-puromycin antibody from EMD Millipore Co. (Merck Millipore). Penicillin, streptomycin, fetal bovine serum (FBS) and trypsin were purchased from GIBCO Life Technologies Inc., EMEM (LM007-75 and LM007-87 for glucose starvation and normal glucose condition, respectively) and DMEM (LM001-56) media from Welgene Biotech Co. Each media contains same concentrations of ingredients except glucose. Media for glucose-starved condition, low-glucose condition and normal glucose condition contain 0, 500 and 1000 mg/L (0, 2.8 and 5.5 mM) of glucose, respectively.

Primary and secondary antibodies used in the immunoblotting are as follows: monoclonal anti-MBP antibody (New England Biolabs, E8032S, 1:10,000 dilution), monoclonal, horseradish peroxidase (HRP)–conjugated, anti-GST antibody (B-14) (Santa Cruz Biotechnology, sc-138 HRP, 1:1000), monoclonal anti-Myc antibody (Cell Signaling Technology, 71D10, 1:3000 dilution), monoclonal, HRP-conjugated, anti-GFP antibody (Miltenyi Biotec, 130-091-833, 1:3000 dilution), monoclonal anti–α-tubulin antibody (Santa Cruz Biotechnology, sc-5286 HRP, 1:1000), anti-mouse immunoglobulin G (IgG)–peroxidase antibody (Sigma-Aldrich, A9044, 1:5000), and anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, 7074, 1:5000 dilution). Signal detection was performed with the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific).

Polyclonal anti-Sprouty2 antibody was obtained from Sigma. The monoclonal anti-phospho-ERK1/2 (Thr202/Tyr204) and polyclonal anti-phospho-CREB, anti-CREB, anti-ERK1/2, anti-phospho-AKT (Ser473) and anti-AKT antibodies were obtained from Cell Signaling (Danvers, MA). The polyclonal anti-COX-2 antibody was obtained from Abcam (Cambridge, MA). The monoclonal anti-α-tubulin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG were obtained from Bio-Rad Laboratories (Hercules, CA). Human chorionic gonadotropin (hCG), 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) and LY294002 were obtained from Sigma. Recombinant human amphiregulin was obtained from R&D systems (Minneapolis, MN). Wortmannin, PD98059 and U0126 were obtained from Calbiochem (San Diego, CA).

All chemicals were purchased from Sigma Chemicals (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA) unless specified otherwise. Cell culture reagents were purchased from Mediatech, Inc. (Herndon, VA). The monoclonal anti-CaR antibody used in this study was described earlier [23 (link)]. The monoclonal anti-α-tubulin antibody, and the polyclonal anti-p115 and anti-calpain 1 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The monoclonal anti-calpain-1/2 (small subunit) antibody was purchased from EMD Chemicals (San Diego, CA). The anti-filamin A antibodies were generated (in rabbit) against two synthetic peptides representing human filamin A in the first hinge region (AA^1755–1765) and in the C-terminus (AA^2635–2647) (Figure 2B). Transfection of synthetic siRNAs (negative control and CaR-specific) and establishment of stable cell lines with silenced p115 were described earlier [26 (link)]. The goat anti-mouse or rabbit Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies were obtained from Molecular Probes, Inc. (Eugene, OR). The SuperSignal West Pico chemiluminescent substrate and BCA protein assay reagent were obtained from Pierce (Rockford, IL).