Total protein was collected using the RIPA (Beyotime, China) and protease inhibitor cocktail (Roche, Switzerland). Standard Western blotting procedure[15 (link)] was followed with PVDF membrane (Bio-Rad, USA) used for protein transfer. Detection of HRP conjugated secondary antibody was performed with ECL (Vazyme, China). The antibodies used were as follows: rabbit anti-PUM1 (1:1000 dilution; Abcam), mouse anti-Actin (1:5000 dilution; Sigma, USA), rabbit anti-CDKN1B (1:1000 dilution; CST, USA), mouse anti-GAPDH (1:2000 dilution; Proteintech, USA), and mouse anti-α-Tubulin (1:5000 dilution; Santa Cruz, USA). The band intensity of specific proteins was quantified after normalization with that of α-Tubulin or GAPDH.
Mouse anti α tubulin
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Germany, Canada
Mouse anti-α-tubulin is a primary antibody that recognizes the α-tubulin subunit of the tubulin protein. Tubulin is a key structural component of microtubules, which are essential for various cellular processes such as cell division, intracellular transport, and cell motility. This antibody can be used for the detection and localization of α-tubulin in biological samples through techniques like immunofluorescence or Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti β actin, by Merck Group (5 mentions)
Goat anti vegfr2, by R&D Systems (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Mouse anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Mouse anti flag, by Merck Group (2 mentions)
Ecl detection system, by GE Healthcare (2 mentions)
Mouse anti cd63, by Abcam (2 mentions)
Mouse anti eea1, by BD (2 mentions)
Ecl system, by Thermo Fisher Scientific (2 mentions)
Recombinant human vegf a165, by Genentech (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Rabbit anti oct4, by Abcam (2 mentions)
Mouse anti lama c, by Santa Cruz Biotechnology (2 mentions)
Mouse anti sox2, by Abcam (2 mentions)
Protein a g magnetic bead, by Thermo Fisher Scientific (2 mentions)
Mouse anti phospho p42 44, by Cell Signaling Technology (2 mentions)
53 protocols using mouse anti α tubulin
1
Western Blot Analysis of Protein Expression
2
AKT and HEY Signaling in CVP Cells
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CVPs were cultured in basal medium in the presence or absence of endothelin-1 growth factor for 5 days. For the preparation of cell lysates, cell pellets were incubated in RIPA lysis buffer (1% Nonidet P-40, 0.5% deoxycholate, 5 M NaCl and 1 M Tris (pH 7.4)) for 10 min at 4 °C, and then centrifuged at 11,400g for 15 min at 4 °C. Quantification of protein extract was carried out using the Protein Assay (Bio-Rad) according to the manufacturer's instructions. Electrophoretic analysis was performed using 10–15% SDS–PAGE gel (Bio-Rad). Gels were blotted onto nitrocellulose membrane (Amersham Biosciences), which were then probed with rabbit anti-human AKT, anti Phospho-AKT (Ser473; Cell Signaling), mouse anti β-actin (Santa Cruz), rabbit anti-human HEY1 and HEY2 (both from GeneTex) and mouse anti α-tubulin (Santa Cruz) primary antibodies according to the manufacturer's instructions. Primary antibodies were detected with species-specific horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) and ECL western blotting detection system (Amersham Biosciences).
3
Gene Expression and Protein Analysis in Skin
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RNA from head skins was extracted using 0.5 ml of Trizol reagent (Invitrogen) according to the manufacturer’s protocol. RT-PCR was performed according to standard protocols using a reverse transcriptase kit and random primers (Promega). PCR products were size-fractionated by 2% agarose gel electrophoresis. Realtime PCR was performed in triplicate with Power SYBR Green PCR Master Mix on a 7500 Real-Time PCR Detection System (Applied Biosystems). Relative mRNA expression levels were normalized to those of GAPDH and analyzed using the 2-DDCt method. All gene-specific primers were designed by Primer 5 Software. Primer sequences are depicted in Supplementary Table 1 .
Dissected head skins were placed in RAPI protein lysis solution (Byotime, China) and lysed on ice for 10 minutes with a Micro Tissue Grinder. Samples were separated by 10% SDS-PAGE and western blots were prepared by standard procedures using either goat anti-TYR (1: 500, Santa Cruz Biotech), mouse anti-β-galactosidase (1: 800, Promega), mouse anti-TYRP1 (1: 500, Abcam) or rabbit anti-EDN3 (1: 1000, Abcam) as primary antibodies and appropriate secondary IRDye® 700CW or IRDye® 800CW-conjugated antibodies. Protein bands were scanned using a LI-COR machine. Mouse anti-α-Tubulin or anti-β-Actin antibodies (1: 2000, Santa Cruz Biotech) were used as internal controls. Protein bands were quantified using Image J software.
Dissected head skins were placed in RAPI protein lysis solution (Byotime, China) and lysed on ice for 10 minutes with a Micro Tissue Grinder. Samples were separated by 10% SDS-PAGE and western blots were prepared by standard procedures using either goat anti-TYR (1: 500, Santa Cruz Biotech), mouse anti-β-galactosidase (1: 800, Promega), mouse anti-TYRP1 (1: 500, Abcam) or rabbit anti-EDN3 (1: 1000, Abcam) as primary antibodies and appropriate secondary IRDye® 700CW or IRDye® 800CW-conjugated antibodies. Protein bands were scanned using a LI-COR machine. Mouse anti-α-Tubulin or anti-β-Actin antibodies (1: 2000, Santa Cruz Biotech) were used as internal controls. Protein bands were quantified using Image J software.
4
Western Blot Analysis of ACE, ACE2, and TMPRSS2
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Total cellular protein was extracted by dissolving cells in RIPA buffer. Protein concentration was measured using the Pierce BCA protein assay kit (23225, Thermo Fisher). Samples with normalized total protein concentrations were denatured at 95○C for 5 min and separated by SDS-PAGE, and then transferred to a PVDF membrane and blocked in 5 % skimmed milk in PBST for 1 h. Rabbit anti-ACE (1:1000, PA5–83080, Thermo Fisher), rabbit anti-ACE2 (1:1000, ab108252, Abcam), rabbit anti-TMPRSS2 (1:1000, ab92323, Abcam), mouse anti-β-actin (1:5000, Sigma) or mouse anti-α-tubulin (1:5000, Santa Cruz) in blocking buffer were added and incubated overnight at 4 oC. After washing with PBST, the membranes were incubated with anti-rabbit or anti-mouse secondary antibody conjugated with horse-radish peroxidase (1:5000, GE Healthcare) for 1 hr and washed. Membranes were developed using a chemiluminescence kit (Advansta, Griffin Biotech). The normalization of protein loading was confirmed with β-actin or α-tubulin expression.
5
Immunostaining of Cytoskeleton Proteins
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The following primary antibodies were used: mouse anti–γ-tubulin (1:500; Sigma-Aldrich, St. Louis, MO, United States), mouse anti–α-tubulin (1:500; Santa Cruz Biotechnology, Dallsa, TX, United States), mouse anti-GFP (1:500; Roche, Basel, Switzerland), rabbit anti-GFP (1:500; Abcam, Cambridge, United Kingdom), mouse 22C10 (1:200; DSHB), mouse 21A6 (1:200; DSHB), iFluorTM 555 Phalloidin (1:200; Yeasen, Shanghai, China). The following secondary antibodies were used: goat anti–mouse Alexa Fluor 488 or Alexa Fluor 594 or goat anti–rabbit Alexa Fluor 488 or Alexa Fluor 594.
6
Western Blot Analysis of B7-H4 and Cell Markers
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Cells were lysed in M-PER lysis buffer (ThermoFisher) and processed for Western blot as described in [26 (link)]. Primary antibodies used were rabbit anti-B7-H4 (1:500, D1M81, Cell Signaling, Danvers, MA, USA), mouse anti-Flag (1:500, MAB3118, Sigma-Aldrich), mouse anti-GAPDH (1:500, 6C5, Santa Cruz Technology, Dallas, TX, USA), and mouse anti-α-Tubulin (1:200, B-7, Santa Cruz Technology). Secondary antibodies were IRDye 680RD and 800CW Goat anti-Mouse and Goat anti-Rabbit (LI-COR, Lincoln, NE, USA). Odyssey CLx (Li-Cor®) Image Studio v4.0.21 software was used to visualize fluorescence signals on the membranes.
7
PODXL Protein Expression in HEK293 and HeLa Cells
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HEK293 cells and HeLa cells were cultured in high-glucose DMEM media with 1% antibiotics. The media was changed to high-glucose DMEM without antibiotics for 1 day before transfection. The GeneINTM transfection reagent GST-1002 was used to transfect cells with wild-type, M1I and W341X PODXL cDNAs cloned into a pcDNA3.1+ vector. Forty-eight hours after transfection, the cells were lysed with RIPA buffer, separated on an 8% SDS-PAGE gel, transferred, and probed using antibodies diluted in 5% skim milk. The antibodies used were goat anti-PODXL (R&D, AF1658, 1:2500), mouse anti-α-tubulin (Santa Cruz, sc-8035, 1:20 000), rabbit anti-goat IgG conjugated to HRP (Abcam, Cambridge, UK, ab6741, 1:5000), and goat anti-mouse IgG conjugated to HRP (Bio-Rad, Hercules, CA, USA, 170-6516, 1:5000).
8
Immunoblotting Analysis of Cytoskeletal and Immune Signaling Proteins
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Rabbit anti-cytoskeletal actin (Bethyl Laboratories, Montgomery, TX, USA), mouse anti-CVB3 VP1 (Dako, Copenhagen, Denmark), mouse anti-α tubulin (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-STING, rabbit anti-phospho-STING (Ser336), rabbit anti-TBK-1, rabbit anti-phospho-TBK-1 (Ser172), rabbit anti-IRF-3, and rabbit anti-phospho-IRF-3 (Ser396) (all from Cell Signaling Technologies, Danvers, MA, USA) antibodies were used. The enhanced chemiluminescence substrate femto LUCENT™ PLUS-HRP (G-Biosciences, St. Louis, MO, USA) was applied, and images of bands were captured using an Image Quant™ LAS 4000 Mini system (GE Healthcare Life Sciences, Little Chalfont, UK). Quantification of band densities was performed using ImageJ software (NIH, Bethesda, MD, USA).
9
Western Blot Analysis of Protein Lysates
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Protein lysates were collected in RIPA buffer (50 mmol/l Tris pH 7.4, 1%
NP-40, 0.25% sodium deoxycholate, 150 mmol/l NaCl, 1 mmol/l EDTA),
1 mmol/l phenylmethylsulfonyl fluoride and protease inhibitor cocktail
(Roche), stored at −80 °C (30 min), defrosted on ice and
centrifuged at 4 °C (10 min,
11 269g ). The supernatant
was recovered and total protein concentration was assessed by Bio-Rad assay.
Total proteins (25 μg) were resolved on a 12% SDS–PAGE gel and
transferred onto a nitrocellulose membrane. Primary antibodies used were mouse
anti-p65 (Santa Cruz Sc-8008), mouse anti-β-actin (Sigma–Aldrich
A-5441), mouse anti-α-tubulin (Santa Cruz Sc-5286) and rabbit
anti-11β-HSD1 (Ricketts et
al. 1998 ).
Secondary antibodies (Dako, Ely, Cambridgeshire, UK) anti-mouse and anti-rabbit
conjugated with HRP were added at a dilution of 1/5000. Equal loading of protein
content was verified using β-actin and the bands were visualised using ECL
detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
NP-40, 0.25% sodium deoxycholate, 150 mmol/l NaCl, 1 mmol/l EDTA),
1 mmol/l phenylmethylsulfonyl fluoride and protease inhibitor cocktail
(Roche), stored at −80 °C (30 min), defrosted on ice and
centrifuged at 4 °C (10 min,
11 269
was recovered and total protein concentration was assessed by Bio-Rad assay.
Total proteins (25 μg) were resolved on a 12% SDS–PAGE gel and
transferred onto a nitrocellulose membrane. Primary antibodies used were mouse
anti-p65 (Santa Cruz Sc-8008), mouse anti-β-actin (Sigma–Aldrich
A-5441), mouse anti-α-tubulin (Santa Cruz Sc-5286) and rabbit
anti-11β-HSD1 (
al. 1998
Secondary antibodies (Dako, Ely, Cambridgeshire, UK) anti-mouse and anti-rabbit
conjugated with HRP were added at a dilution of 1/5000. Equal loading of protein
content was verified using β-actin and the bands were visualised using ECL
detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
10
Western Blot Analysis of STC-2 Protein
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Cells transfected with miR-184 mimic/inhibitor or negative controls for 48 h were washed with three changes of PBS. After removing PBS, cells were immediately frozen with liquid N2. Cold 1 × Pierce RIPA Buffer (Thermo Scientific, #89,900, US) containing 1 × HALT Phosphatase Inhibitor Cocktail (Thermo Scientific, #78420, US) and 1 × HALT Phosphatase Inhibitor Cocktail (Thermo Scientific, #78430, US) were added immediately for cell lysis. Cells were scraped from the plastic surface and collected in Protein LoBind Tubes. After 10 min incubation on ice, cell lysates were centrifuged (13000 g, 4 °C, 30 min), and supernatants containing proteins were acquired. Protein concentration was measured using the Pierce BCA Assay Kit (Thermo Scientific, #23227, US) following the manufacturer’s protocol. Samples were blotted to the NC membrane (GE Healthcare Life Science, #10600114, Germany) after electrophoresis. The blotted membrane was then blocked using EveryBlot Blocking Buffer (BioRad, #12010020, US). The antibodies used for detection were rabbit anti-STC-2 (Abcam, #ab63057, US), and mouse anti-α-tubulin (Santa Cruz, #SC-5286, US), goat-anti-rabbit-HRP (Genetex, #GTX213110-01, US), and goat-anti-mouse-HRP(Genetex, #GTX213111-01, US) diluted to the manufacturer’s recommended concentration.
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