Hrp conjugated β actin antibody

Protein lysates were collected using Blue Lysis Buffer and analyzed by Western blot as previously described [22 (link)]. The recipe for Blue Lysis Buffer consists of 25 mL 2x Novex Tris-Glycine Sample Loading Buffer SDS (ThermoFisher Scientific, Waltham, MA USA, Cat# LC2676), 20 mL T-PER Tissue Protein Extraction Reagent (ThermoFisher Scientific, Waltham, MA USA, Cat# 78510), 200 µL 0.5 M EDTA pH 8.0, 2–3 complete Protease Cocktail tablets for 50 mL, 80 µL 0.1 M Na₃VO₄, 400 µL 0.1 M NaF, 1.3 mL 1 M dithiothreitol. Briefly, primary antibodies against capsid of Venezuelan equine encephalitis virus, TC-83 (Subtype IA/B) Capsid (antiserum, Goat) (BEI resources, NR-9403), VEEV GP (antiserum, Goat), VEEV nsP2 (Kerafast, Boston, MA, USA, Cat# 8A4B3), PERK (C33E10) (Cell Signaling, Danvers, MA, USA, Cat# 3192S), or horse radish peroxidase (HRP)-conjugated β-actin antibody (Abcam, Cambridge, MA, USA, Cat# ab49900) were diluted in 3% milk solution per the manufacturer’s recommended dilutions followed by the addition of the appropriate secondary antibody either anti-rabbit HRP-conjugated (Cell Signaling, Danvers, MA, USA, Cat# 7074), or anti-goat HRP-conjugated antibody. PDVF membranes were imaged on a Chemidoc XRS molecular imager (Bio-Rad, Hercules, CA, USA) using the SuperSignal West Femto Maximum Sensitivity Substrate kit (ThermoFisher, Scientific, Waltham, MA, USA, Cat# 34095).

Whole cell or mitochondrial lysates from differentiated C2C12 cells were prepared with RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 0.5 mM EDTA, 150 mM sodium chloride) supplemented with protease inhibitor cocktail (Roche). Forty μg of lysate, 20 μg of mitochondrial pellet, or 10 μg of supernatant were run on a 10% gel (Bio-Rad) and transferred to PVDF membrane (Immobilon). The membrane was probed with rabbit polyclonal anti-NMNAT1 (1:500 dilution) as previously described (Zhang et al., 2009 (link)) or anti-VDAC (Abcam) followed by secondary antibody incubation. Immunoblots were developed using SuperSignal West femto kit (Thermo Fisher Scientific) on a Bio-Rad imaging system. Blots were then stripped and re-probed with HRP-conjugated β-actin antibody (Abcam).