Anti β actin antibody c4

Briefly, proteins from cell lysates were separated on 12% (w/v) SDS-PAGE and transferred onto a PVDF membrane (Millipore, IPVH00010). The membrane was blocked in 0.3% (w/v) nonfat dry milk, TBST (1x TBS, 0.1% Tween-20) as blocking buffer, and then incubated with the primary antibody in Can Get Signal Immunoreaction Enhancer Solution I (TOYOBO, NKB201) as diluent at 4°C overnight. After washing with TBST, the membrane was incubated with the horseradish peroxidase-conjugated secondary antibody in Can Get Signal Immunoreaction Enhancer Solution II (TOYOBO, NKB2301) as diluent at room temperature overnight. Bands were visualized using a chemiluminescent detection system (ATTO, WSE-6100H-CP LuminoGraph I). Primary antibodies used in this study are listed below: anti-AKT antibody (B-1) (diluted at 1:1000, Santa Cruz, sc-5298), anti-phospho AKT1/2/3 antibody (11E6) (diluted at 1:1000, Santa Cruz, sc-81433), anti-STAT3 antibody (D3Z2G) (diluted at 1:5000, Cell Signaling Technology, 12640S), anti-phospho STAT3 antibody (D3A7) (diluted at 1:5000, Cell Signaling Technology, 9145S), anti-β-Actin antibody (C-4) (diluted at 1:5000, Santa Cruz, sc-47778). Secondary antibodies used in this study are listed below: anti-IgG (H + L chain) (Mouse) pAb-HRP (diluted at 1:10000, MBL, 330), anti-IgG (H + L chain) (Rabbit) pAb-HRP (diluted at 1:10000, MBL, 458).

hARF6 ORF cDNA lentiviral clone was obtained from GeneCopoeia (EX-OL00097-LX304-B).
DSS was from Cayman Chemical. Puromycin, hexadimethrine bromide (polybrene), Lucifer Yellow CH dilithium salt, and fluorescein isothiocyanate–dextran were purchased from Sigma. EZ-Link Sulfo-NHS-Biotin and Collagenase, Type I, were obtained from Thermo Fisher Scientific. Polyethylenimine (PEI) was from Polysciences. AGK2 was from MCE. TM, TM-P4-Thal, and IMP-1088 were synthesized as previously described (29 (link), 38 (link), 39 (link)).
Antibodies were obtained from the following sources: Cell Signaling Technology—rabbit anti-E-cadherin (24E10), E-cadherin (24E10) rabbit mAb (Alexa Fluor® 488 Conjugate), rabbit anti-SirT2 (D4050), rabbit anti-HSP90 (C45G5), rabbit anti-Arf6 (D12G6), anti-mouse IgG, HRP-linked antibody (#7076), anti-rabbit IgG, and HRP-linked antibody (#7074); Santa Cruz—anti-β-actin antibody (C4) and anti-GAPDH (0411).

After cell lysis using RIPA buffer (Sigma) containing a protease inhibitor cocktail (GenDEPOT), cell lysates were mixed with lane marker reducing sample buffer (Thermo Fisher Scientific) and boiled. The proteins were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked for 1 h at room temperature (RT) with 5% skim milk prepared in TBS-T buffer. The membrane was then incubated overnight at 4 °C with a primary antibody diluted 1:1000 in 5% skim milk. After washing in TBS-T buffer, the membrane was incubated for 1 h at RT with an HRP-conjugated secondary antibody diluted 1:5000 in 5% skim milk. After washing again, signals were detected using West-Q Pico ECL solution or West-Q Femto clean ECL solution (GenDEPOT). An anti-HDAC6 antibody (D21B10, Cell Signaling Biotechnology) and an anti-β-actin antibody (C4, Santa Cruz Biotechnology) were used as the primary antibodies. HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG were used as the secondary antibodies.

Anti-C. elegans LMP-1, HSP-60, CYP-33, and SQV-8 monoclonal antibodies were purchased from Developmental Studies Hybridoma Bank (DSHB). Those antibodies were originally generated by Dr. Michael L. Nonet’s lab (Hadwiger et al., 2010 (link)). Anti-β-actin antibody (C4) was purchased from Santa Cruz (sc-47778).