Standard western plotting protocols were followed. Recombinant Anti-ACE2 antibodies (Cat# ab108209; Abcam: N-terminal) and (Cat# ab15348; Abcam: C-terminal), alpha-tubulin (Cat# sc-32293; Santa-Cruz) and beta-actin (Cat# sc-69879; Santa-Cruz) were used for ACE2/dACE2, alpha-tubulin detection and beta-actin, respectively.
Alpha tubulin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Alpha-tubulin is a core component of microtubules, which are structural elements found in the cytoskeleton of eukaryotic cells. Alpha-tubulin, along with beta-tubulin, forms the tubulin heterodimer, the building block of microtubules. Microtubules play a crucial role in various cellular processes, including cell division, intracellular transport, and the maintenance of cell shape and structure.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Santa Cruz Biotechnology (5 mentions)
β actin, by Merck Group (3 mentions)
P70s6k1 t389, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Fortis Life Sciences (2 mentions)
Anti redd1, by Proteintech (2 mentions)
Ecl plus reagent, by Bio-Rad (2 mentions)
Flag m2, by Merck Group (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Ferritin light chain, by Santa Cruz Biotechnology (2 mentions)
Odyssey clx ir scanner, by GraphPad (2 mentions)
Prism 7, by GraphPad (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Tu 02, by Santa Cruz Biotechnology (1 mentions)
Ecl reagent, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Chemiluminescence imaging system, by Syngene (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
26 protocols using alpha tubulin
1
Western Blotting of ACE2 and Cytoskeletal Proteins
2
Quantitative Western Blot Analysis
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Cell lysates were isolated by RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton x-100, 1% Na-Doc, 0.1% SDS) supplemented with protease inhibitor cocktail (Roche). Cell extracts were quantitated using a BCA protein assay kit (Thermo). Western blot analysis was performed standard techniques for Twist1 (Abcam, ab50887, 1:1000, Cambridge, MA), Prrx1 (Origene, TA803116, 1:1000, Rockville, MD, USA), TNC (Genetex, GTX62552, 1:1000, USA) and alpha-tubulin (Santa Cruz, TU-02, 1:3000, Dallas, TX). Protein detection for Western blotting was performed using ECL reagent (#1705061, Bio-Rad). The antibodies are described in Supplementary Table 1 . Uncropped images of the gels are shown in Supplementary Fig. 11 .
3
Protein Blotting Antibody Validation
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Antibodies used for protein blotting were TBL1XR1 (Novus), HDAC3 and NCoR (Abcam) and alpha-tubulin (Santa Cruz). Horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad) were used to detect primary antibodies.
4
Western Blot Analysis of Cellular Proteins
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Total cell lysates prepared from treated and untreated cells were harvested at defined time-points and subjected to Western blotting analysis as previously described [30 (link),32 (link),41 (link)]. Primary antibodies were purchased from commercial vendors and used for detecting human HuR, p27, BCL-2, alpha-tubulin (Santa Cruz Biotechnology, Dallas, TX, USA), cyclin D1, cyclin E1, HIF1-α, MITF, VEGF-A, Caspase-9, and PARP (Cell Signaling Technology Inc., Beverly, MA, USA) and beta-actin (Sigma Aldrich, St. Louis, MO, USA). Protein bands were detected using appropriate horseradish peroxidase (HRP)-tagged secondary antibodies (Santa Cruz Biotechnology) and an enhanced chemiluminescence kit (Thermo Scientific, MA, USA). Protein expression levels were detected on a chemiluminescence imaging system (Syngene, Frederick, MD, USA), and the relative protein expression compared to beta-actin or alpha-tubulin was quantified using Gene Tools software (Syngene) as previously described [30 (link),32 (link),41 (link)]. Experiments were repeated at least three separate times for reproducibility, and the data were analyzed for statistical significance.
5
Mitochondrial Dynamics Protein Analysis
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Primary antibodies used for Western Blotting were anti-GAPDH (MAB374; Millipore), VDAC1 (ab14734; Abcam), MFN1 (ab57602; Abcam), MFN2 (ab56889; Abcam), OPA1 (612607; BD Biosciences), FIS1 (10956-1-AP; Proteintech), MFF (17090-1-AP; Proteintech), DRP1 (Dlp1; 611112; BD Biosciences), involucrin (I9018; Sigma), profilaggrin (sc66192; Santa Cruz), HSP70 (ADI-SPA-810; Enzo life science), alpha-tubulin (sc8035; Santa Cruz). The following secondary antibodies were used: horseradish peroxidase-conjugated rabbit anti-mouse (P0161; DAKO) and horseradish peroxidase-conjugated goat anti-rabbit (PO448; DAKO). Hepes, D-Mannitol, Sucrose, Ethylenediaminetetraacetic acid (EDTA), Trizma Hydrochloride (Tris-HCL), Sodium chloride (NaCl), Igepal (NP-40), Sodium deoxycholate (DOC), Sodium dodecyl sulfate (SDS), Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were purchased from Sigma Aldrich.
6
Cellular Compartment Fractionation
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Subcellular fractionation of the cytoplasm, mitochondria and nucleus was achieved using a fractionation kit (Abcam, Burlingame, CA, USA) according to the manufacturer's recommendations. Alpha‐tubulin (Santa Cruz Biotechnology, #sc‐8035), COX IV (Abcam, #ab14744) and Lamin A (Santa Cruz Biotechnology, #sc‐6214) were used as endogenous control markers for the cytoplasm, mitochondria and nucleus, respectively.
7
Immunoblotting and Immunofluorescence Protocols
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The sources and catalog numbers of the antibodies used were as follows: PC1 (sc-130554), c-MYC (sc-40), HA (sc-7392), AXIN1 (sc-293190), β-catenin (sc-7963), p-TAZ (Ser89; sc-17610), and alpha tubulin (sc-5286; all from Santa Cruz Biotechnology); HA (ab969110), Myc (ab9106), and Flag (ab1257; all from Abcam); YAP/TAZ (no. 8418), α-tubulin (no. 2144), AXIN1 (no. 2087), c-MYC (no. 9402), phospho-CREB (no. 9198), Histone H3 (no. 60538), and LSD1 (Lysine-specific demethylase 1; no. 2139; all from Cell Signaling); β-actin (A2228; Sigma); β-catenin (no. 610153) and TAZ (no. 560235; both from BD Biosciences); and anti–active-β-catenin (ABC 05–665; Millipore). For immunofluorescence, anti-HA tag antibody (ab18181; Abcam) and anti-Myc tag rabbit (sc-2278; Cell Signaling) were used.
8
Spinal Cord Protein Expression Analysis
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The animals were deeply anesthetized with 3% isoflurane via a nose cone for approximately 2 to 3 min, after which L4∼L5 spinal cord was quickly removed and immediately frozen in liquid nitrogen and stored at −70℃ until tissue lysis. The extracted proteins were heated at 95℃ for 5 min to denature the 3D structure of the protein and separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. With the help of an I-Blot transfer machine (Invitrogen, Waltham, MA, USA), the proteins were transferred to PVDF membranes and treated for 7 min. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween (TBST) and incubated overnight with primary antibody in TBST buffer at 4℃. The primary antibodies used were against MMP-2 (1:100, Santa Cruz Biotech, Dallas, TX, USA), MMP-9 (1:100, Santa Cruz Biotech, Dallas, TX, USA), TIMP-2 (1:100, Santa Cruz Biotech, Dallas, TX, USA) and alpha-tubulin (1:10000, Santa Cruz Biotech, TX, USA). The next day, the membranes were washed with TBST buffer several times and incubated with secondary antibody for an hour at room temperature. The horseradish peroxidase activity was measured using an enhanced chemiluminescence kit (ECL kit, Pierce, Thermoscientific, Rockford, IL, USA). The chemiluminescent signals were captured on autoradiography film.
9
Western Blot Analysis of REDD1 and p70S6K1
10
Western Blot Analysis Protocol
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To extract protein, cells were treated in cold RIPA buffer containing protease inhibitor. To denature proteins, lysates were added to 4× loading buffer and heated to 95 °C for 10 min. Total cell lysate (30–50 µg) were loaded onto SDS PAGE gels, and then transferred to PVDF membranes. Blots were incubated with primary antibodies overnight at 4 °C, followed by detection with secondary antibody. The antibodies used were: MECOM (Cell Signaling Technology, #C50E12, 1:500 dilution in PBST), beta-Actin (Cell Signaling Technology, #4967, 1:1000 dilution in PBST), KDR/VEGFR2 (Santa Cruz, #sc-6251, 1:500 dilution in PBST), GAPDH (Santa Cruz, #sc-32233, 1:1000 dilution in PBST), alpha Tubulin (Santa Cruz, #sc-5286, 1:1000 dilution in PBST), AP1(Signaling Technology, #9165, 1:1000 dilution in PBST), goat anti-rabbit HRP-conjugated antibody (Jackson Labs, #111-035-144, 1:5000 dilution in PBST).
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