Anti mouse igg hrp

After treatment, cells were lysed in cell lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich). The total cell lysates were loaded on SDS-PAGE and electro-transferred into nitrocellulose membrane, followed by incubation with the appropriate primary antibody at 4 °C overnight. The primary antibodies used were mouse anti-cyclophilin A (ab-58144) (1:1000), mouse anti-β Actin (sc-47778) (1:1000), mouse anti-CD 47 antibody (1:1000) (B6H12.2, Novus Biologicals) and rabbit anti-calreticulin (1:1000) (ab-2907). The membranes were later incubated with specific secondary antibodies: anti-mouse IgG-HRP (Abcam) at a dilution of 1:5000. The proteins were visualized with Clarity Western ECL substrate. The bands were analyzed by Quantity One 1D image-analysis software (Bio-Rad Laboratories, Hercules, CA, USA).

For immunohistochemistry and immunofluorescence, kidney slices were placed in OCT, and sections were processed with standard procedures. For immunoblotting, kidneys were homogenized in RIPA buffer containing protease inhibitor cocktail (Roche). After quantification with BCA, equal amount of proteins (30−100 µg) were loaded onto an SDS-PAGE gel, followed by standard immunoblotting. Band densities were quantified with Image J (NIH). Primary antibodies used were AMPKα (Cell Signaling Technology, 2532, 1:1000), phospho-AMPKα (Thr172) (Cell Signaling Technology, 2531, 1:1000), p44/42 MAPK (Erk1/2) (Cell Signaling Technology, 4695, 1:500) and phospho-p44/42 MAPK (Erk1/2) (Cell Signaling Technology, 4370, 1:500), anti-Catalase (R&D, AF3398, 1:50) and Ki-67 (abcam, ab16667, 1:200). Secondary antibodies were anti-rabbit IgG-HRP (Santa Cruz Biotechnology, sc-2357, 1:5000), anti-mouse IgG-HRP (Abcam; ab97046, 1:5000), Dolichos Biflorus Agglutinin (DBA) Fluorescein (Vector lab, FL-1031, 1:50), Lotus Tetragonolobus Lectin (LTL) Fluorescein (Vector labs, FL-1321-2, 1:50), donkey-anti-rabbit IgG Alexa Fluor 647 (Invitrogen, A-31573, 1:200), donkey-anti-goat IgG Alexa Fluor 555 (Invitrogen, A-21432, 1:200).

The content of free and polymerized tubulin in HL-1 and N2a cells was assessed using a “Microtubules/Tubulin in vivo Assay “kit (Cytoskeleton Inc.) in accordance with the manufacturer’s manual. Cells were homogenized in cell lysis and microtubule stabilization buffer (100 mM PIPES pH 6.9, 5 mM MgCl₂, 1 mM EGTA, 30% (v/v) glycerol, 0.1% Nonidet P40, 0.1% Triton X-100, 0.1% β-mercaptoethanol, 0.001% antifoam) supplemented with 0.1 mM GTP, 1 mM ATP and protease inhibitor cocktail. In addition, cell fractions containing 10 μM taxol and 2 mM CaCl₂ were used as the positive and negative controls. Lysates were centrifuged at 2000 x g for 5 min at 37 °C to remove intact cells. Supernatants were centrifuged at 100000 x g for 30 min at 37 °C to separate microtubules from soluble (free) tubulin. The pellets containing polymerized tubulin were suspended in ice-cold 2 mM CaCl₂.
Free tubulin and polymerized tubulin fractions were loaded on 10% polyacrylamide gels. Proteins were transferred using the Trans-Blot SD Semi-Dry Transfer Cell (BioRad). Blots were blocked in 5% nonfat milk and probed with anti Tubb2A (ab170931) antibody for 2 h at room temperature. Immunoblots were incubated with secondary antibodies (anti-mouse IgG, HRP, Abcam) for 1 h at room temperature. Detection was conducted using a chemiluminescence kit (Pierce ECL Western Blotting Substrate).

The quantity of Bax and Bcl-xL proteins in ponatinib-treated cells were analyzed by Western blot. After the treatment, cells were washed in PBS and centrifuged at 200× g for 5 min at 20 °C. Subsequently, cells were lysed (1% Triton X-100 in PBS supplemented with protease- and tyrosine phosphatase inhibitor) and sonicated. Cell-lysate samples were incubated in a buffer (62.5 mM Tris, 2% SDS, 10% glycerol, 5% MEA, pH 6.8) for 5 min at 95 °C. The separation of proteins was performed by SDS-polyacrylamide gel (15%). After Western blotting, the membrane was stained by anti-human Bax, Bcl-xL (Cell Signaling Technology, Danvers, MA, USA) or anti-actin (Abcam, Cambridge, UK). After that, biotinylated anti-rabbit IgG (for Bax and Bcl-xL; Invitrogen, Waltham, MA, USA) or anti-mouse IgG-HRP (for actin; Abcam, Cambridge, UK) secondary antibodies were used. Avidin–biotin complex (ABC) was used for the detection of biotinylated antibody. Protein bands were visualized by enhanced chemiluminescence (ECL, Merck Millipore, Burlington, MA, USA).

Briefly, SARS-CoV-2 S1 (Catalog number 40591-V08H, SinoBiological, China), S2 (Catalog number 40590-V02H, SinoBiological, China), and RBD (Catalog number 40592-V08B, SinoBiological, China) were added to reducing buffer and subjected to 12% PAGE and Western blot analysis using the 1:1000 of the mAbs (stock concentration 1 mg/ml) as primary antibody and anti-mouse IgG HRP (Catalog number ab6789, Abcam, Cambridge, UK) as secondary antibody. Visualization was performed using SuperSignal West Pico PLUS chemiluminescent substrate (Catalog number 34579, ThermoFisher Scientific, Waltham, MA) per the manufacturer’s instructions with the iBright FL1500 Imaging System (ThermoFisher Scientific, Waltham, MA).

The following commercially available antibodies were used at the indicated concentrations for western blot: Anti‐ZFP207 (Santa Cruz Biotechnology, sc‐271943, 1:500), Anti–β‐ACTIN (Sigma‐Aldrich, A5441, 1:2,500), Anti‐OCT3/4 (Santa Cruz Biotechnology, sc8628, 1:2,500), Anti‐Nanog (Santa Cruz Biotechnology, sc‐374001, 1:1,000), Anti‐Sox2 (Santa Cruz Biotechnology, sc‐398254, 1:1,000), Anti‐SFRS11 antibody (Abcam, ab196801, 1:2,000), Goat Anti‐Rabbit IgG H&L (HRP) (Abcam, ab6721, 1:5,000), Goat Anti‐Mouse IgG H&L (HRP) (Abcam, 1:1,000, ab6789), Rabbit Anti‐Goat IgG H&L (HRP) (Abcam, 1:5,000, ab6741), and Rabbit Anti‐ goat IgG (HRP) (Abcam, ab6771, 1:5,000), Anti‐Flag (Sigma, F3165, 1:1,000). In all western blots, β‐ACTIN was used as a loading control (Sigma, A5441, 1:2,500). For IF staining, we used Anti‐SSEA1 (Invitrogen, MA5‐17042, 1:250), Anti‐Nestin (Abcam, ab81462, 1:50), Anti‐Tuj1 (Abcam, ab18207, 1:200), Anti‐Caspase 3 Antibody, active (cleaved) form (MERK, AB3623, 1:100), Goat anti‐mouse IgG AF488 (Invitrogen, A11029, 1:1,000), Goat anti‐rabbit IgG AF568 (Invitrogen, A11011, 1:1,000), Anti‐Mouse IgG HRP (Abcam, ab6789, 1:1,000), and Donkey Anti‐Goat IgG AF594 (Invitrogen, A11058, 1:250).