Molecular imager chemidoc xrs

The procedures for western blot analysis and protein visualization were performed according to our previous studies 52 (link), 54 (link). Primary antibodies included anti-Mettl3 (ab195352, Abcam), anti-LDHA (ABN311 - EMD Millipore), and anti-YTHDF1 (ab99080, Abcam). Anti-α-tubulin (66031-1-Ig, Proteintech) was used as a loading control. The signals were detected by enhanced chemiluminescence using a Chemidoc XRS Molecular Imager (Bio-Rad Laboratories Inc.). Quantity One software (version 4.3.0, Bio-Rad Laboratories, Inc.) was used for densitometric analysis.

Protein was extracted from PC12 cells with lysis buffer containing 1% phenylmethylsulfonyl fluoride and 1% protease inhibitor. Lysates were centrifuged at 12,500 × g for 20 min at 4°C, and the supernatant was collected. The total protein concentration was determined with bicinchoninic acid assays. Equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrically transferred onto a polyvinylidene difluoride membrane, which was subsequently blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h. The membrane was subsequently incubated with primary antibodies against mTOR, phospho-mTOR (Ser2448), Akt, phospho-Akt (Ser473), ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), MEK, phospho-MEK (Ser217/221), cleaved-PARP (Asp214), cleaved-caspase 3 (Asp175), cleaved-caspase 9 (Asp315), or GAPDH overnight at 4°C. After being washed with PBS, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After repeated washes with PBS, proteins were visualized by enhanced chemiluminescence. Images of protein bands were captured, and densitometric measurements of band intensity were performed with a ChemiDoc XRS Molecular Imager (Bio-Rad Laboratories, Hercules, CA, USA).

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).

Immunoblotting was performed as previously reported^{7 (link),10 (link)}. The following antibodies were used for detection: rabbit anti-NAMPT (#A300-372A, Bethyl Laboratories, Inc., 1:20,000), rabbit anti-PARP-1 (#ALX-210-302-R100, Enzo Life Sciences, 1:4000), rabbit anti-CDA (#ab56053, Abcam, dilution 1:500), rabbit anti-BLM (#ab2179, Abcam, dilution 1:5000), rabbit anti-β-actin (#A2066, Sigma-Aldrich, 1:5000), rabbit anti-HSP90 (#ab2928, Abcam, 1:5000), rabbit anti-His-tag (#66,005-I, Proteintech, dilution 1:1000), mouse anti-GAPDH (#G8795 1:1000), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#A9169, Sigma-Aldrich, 1:5000), and HRP-conjugated goat anti-mouse IgG (#A3682, Sigma-Aldrich, 1:5000). Bands were visualized by chemiluminescence (Clarity Western ECL Substrate, Bio-Rad), with a ChemiDoc XRS+ Molecular Imager and Image Lab Software (Bio-Rad).