Western Blotting analysis was performed as described previously 32 (link). Total cell extracts were lysed in cell lysis buffer (Beyotime, P0013), and the lysates were centrifuged at 12,000 rpm for 10 min at 4°C. Samples containing 30 µg protein were loaded on SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk or BSA and probed with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Blots were developed with lumi-light Western blotting substrate detection reagents (Roche, 12015200001). Nuclear- and cytoplasmic-enriched fractions were prepared using Nuclear-Cytosol Extraction Kit (Applygene, P1200), according to manufacturer's instructions. Western Blotting analysis was performed using the anti-p27 (Cell Signaling Technology, 3698S), anti-CREB (Cell Signaling Technology, 9197L), anti-phospho-CREB (Ser133) (Cell Signaling Technology, 9198L), anti-S-phase kinase-associated protein 2 (Skp2) (Cell Signaling Technology, 4313S), anti-CDK2 (Cell Signaling Technology, 2546S), anti-Cyclin E1 (Cell Signaling Technology, 4129S), PARP (Cell Signaling Technology, 9532S), anti-GAPDH (Cell Signaling Technology, 2118L), anti-β-arrestin 1 (Abcam, ab32099), anti-β-arrestin 2 (Abcam, ab54790), anti-tubulin (Beyotime, AT819), anti-cyclin D3 (Beyotime, AC856), anti-p21 (Beyotime, AP021).
Anti tubulin
Manufactured by Beyotime
Sourced in China
Anti-tubulin is a laboratory reagent used to study the structure and function of microtubules, which are essential cytoskeletal components in eukaryotic cells. It binds to tubulin, the protein subunit that makes up microtubules, and inhibits their polymerization or causes their depolymerization.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Anti phospho creb ser133, by Cell Signaling Technology (1 mentions)
Anti cdk2, by Cell Signaling Technology (1 mentions)
Anti cyclin e1, by Cell Signaling Technology (1 mentions)
Anti β arrestin 1, by Abcam (1 mentions)
Anti p21, by Beyotime (1 mentions)
Anti creb, by Cell Signaling Technology (1 mentions)
Anti p27, by Cell Signaling Technology (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
Anti β arrestin2, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Anti swiprosin 1, by Novus Biologicals (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Nitrocellulose membrane, by Pall Corporation (1 mentions)
Celecoxib, by Merck Group (1 mentions)
Anti axin2, by Abcam (1 mentions)
Anti sox2, by Cell Signaling Technology (1 mentions)
Anti survivin, by Cell Signaling Technology (1 mentions)
15 protocols using anti tubulin
1
Comprehensive Western Blotting Analysis
2
Swiprosin-1 Protein Expression Analysis
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The VN was dissected from mouse brainstems and homogenized in T–PER Tissue Protein Extraction Reagent (Pierce) supplemented with protease and phosphatase inhibitors (Merck). Samples were separated on an 8% SDS PAGE and transferred to nitrocellulose membranes (Pall Corporation). The membranes were blocked with 5% bovine serum albumin and blotted with antibodies. Anti-swiprosin-1 (IMGENEX) was used at a concentration of 1:2000, and anti-Tubulin (Beyotime) was used at 1:10000. Protein was visualized using IRDye-conjugated anti-mouse or anti-goat secondary antibodies at 1:5000. An Odyssey Infrared Imaging System (LI-COR) was used to analyses the results.
Each mouse was perfused with saline followed by formalin. The brains were fixed overnight at 4 °C in 4% paraformaldehyde and then treated in 30% sucrose at room temperature for 1 hr. The brains were embedded in paraffin and resectioned into 20-μm-thick coronal sections (bregma −0.8 mm to −8 mm). After deparaffination, endogenous peroxidase activity was blocked by incubating the sections in 1.5% peroxide in methanol for 20 min. The sections were blocked in PBS containing 2% normal goat serum, 1% BSA, and 0.3% Triton X-100 and incubated in Anti-swiprosin-1 antibody (IMGENEX) at 1:200 overnight at 4 °C. Horseradish peroxidase (HRP)-labelled streptavidin and DAB were used to visualize each primary antibody.
Each mouse was perfused with saline followed by formalin. The brains were fixed overnight at 4 °C in 4% paraformaldehyde and then treated in 30% sucrose at room temperature for 1 hr. The brains were embedded in paraffin and resectioned into 20-μm-thick coronal sections (bregma −0.8 mm to −8 mm). After deparaffination, endogenous peroxidase activity was blocked by incubating the sections in 1.5% peroxide in methanol for 20 min. The sections were blocked in PBS containing 2% normal goat serum, 1% BSA, and 0.3% Triton X-100 and incubated in Anti-swiprosin-1 antibody (IMGENEX) at 1:200 overnight at 4 °C. Horseradish peroxidase (HRP)-labelled streptavidin and DAB were used to visualize each primary antibody.
3
Cellular Signaling Pathway Profiling
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Celecoxib, cisplatin, 5-fluorouracil (5-FU) and prostaglandin E2 (PGE2) were purchased from Sigma-Aldrich. Each compound was prepared as 1 mM stock solution in dimethylsulfoxide (DMSO) for dilution into various concentrations. The following mouse or rabbit monoclonal primary antibodies were used: anti-E-cadherin (Abcam), anti-MMP-2 (Abcam), anti-Vementin (Abcam), anti-c-MYC (Abcam), anti-Axin-2 (Abcam), anti-Cyclin-D1 (Abcam), anti- β-catenin (Cell Signaling Technology), anti-Survivin (Cell Signaling Technology), anti-SOX-2 (Cell Signaling Technology), anti-p-GSK-3β (Cell Signaling Technology), anti-GAPDH (Beyotime Biotechnology) and anti-Tubulin (Beyotime Biotechnology). HRP conjugated goat anti-mouse or anti-rabbit secondary antibodies were purchased from Beyotime Biotechnology.
4
Protein Quantification of Cellulose Synthases
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Fresh rice stem tissues at heading stage were ground to a fine powder in liquid nitrogen, and 0.5 g powder was extracted using Plant Total Protein Extraction Kit (Invent Biotech, Beijing, China; SD-008/SN-009) according to the manufacturer’s instructions. The protein content was measured with a BCA protein assay reagent (Beyotime Institute of Biotechnology, Jiangsu, China). The protein level of CESA4, CESA7, and CESA9 was determined by western blot analysis as described previously [24 (link)]. The dilution of anti-CESA4, anti-CESA7, anti-CESA9, and anti-tubulin (Beyotime Institute of Biotechnology, Jiangsu, China) was performed as 1:500, 1:500, 1:500, and 1:1000, respectively.
5
Autophagy Regulation by APY0201, BafA1, and Rapa
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APY0201, bafilomycin A1 (BafA1), and rapamycin (Rapa) were obtained from MedChemExpress (MCE, Shanghai, China) and were dissolved in DMSO (Sigma, USA) for in vitro experiments. APY0201 was dissolved in PEG300 (MCE, Shanghai, China) for animal research in vivo. Anti-LC3A/B (#12741S), CTSD (#2284S), CTSB (#311718T), CDK2 (#2546T), CDK4 (#12790T), cyclin E1 (#4129T), p21 (#2947T), p27 (#3686T), and ATG5 (#12994T) were obtained from Cell Signaling Technology (Danvers, USA). Anti-p62 (#PM045) was obtained from MBL (Tokyo, Japan). Anti-CDK6 (#ab124821) and Anti-Ki67 (#ab15580) were obtained from Abcam (Cambridge, UK). Anti-cyclin D1 (#ab143498) and GAPDH (#60004-1-Ig) were obtained from Proteintech (Wuhan, China). Anti-tubulin (#AF0001) was obtained from Beyotime (Shanghai, China).
6
Morusin-Induced Apoptosis and Cell Cycle Regulation
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Cells treated with morusin were lysated by RIPA (Beyotime) lysis buffer supplemented with a protease inhibitor PMSF (Beyotime) for 30 minutes on ice. Then, the mixtures were centrifuged (12,000 ×g) for 10 minutes at 4 °C for separating the cell debris. Protein concentration was measured by BCA assay (Beyotime). Equal protein contents were boiled for about 5 minutes with loading buffer and separated by SDS-polyacrylamide gels electrophoresis (10% gel), followed by transfer to PVDF membranes (Roche, USA). The PVDF membrane was soaked in blocking buffer for 20 minutes and incubated with the primary antibody at 25 °C for 1–2 h. After three washes with PBS, the membrane was incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit/mouse IgG secondary antibody (Beyotime). Lastly, the membrane was visualized by enhanced chemiluminescence chromogenic substrate. Mouse monoclonal antibody (anti-caspase3, anti-phospho-p38, anti-p38) and rabbit monoclonal antibody (anti-phospho-ERK, anti-ERK, anti-phosphor-JNK, anti-JNK, anti-Bax, anti-Bcl-2, anti-tubulin, anti-cyclin D1, anti-CDK4, anti-CDK6) were purchased from Beyotime.
7
Western Blot Analysis of FTO and NOD1
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Cellular lysates were generated by using 1×SDS-PAGE loading buffer. Proteins were extracted from cells and measured with the BCA Protein Assay kit (Beyotime; P0012S), then subjected to SDS-PAGE (8%) gel and transferred to PVDF (Millipore; ISEQ00010) membranes by semidry blotting (Bio-Rad Trans Blot Turbo System). The membranes were blocked with 5% BSA. Protein was blotted with different antibodies. The antibody against FTO was diluted at 1: 500 (Beyotime; AF6936); The antibody against NOD1 was diluted at 1: 1000 (Customized by Genscript); The antibody against NOD1 of human was diluted at 1: 1000 (Absin; abs115515); Anti-Flag (Beyotime; AF519) and anti-Tubulin (Beyotime; AT819) monoclonal antibody were diluted at 1: 2,000; and HRP-conjugated anti-rabbit IgG (Abbkine; A25022) or anti-mouse IgG (Abbkine; A25012) at 1: 5,000. The results were the representative of three independent experiments. The immunoreactive proteins were detected by using WesternBrightTM ECL (Advansta). The digital imaging was performed with a cold CCD camera.
8
Protein Extraction and Western Blot Analysis
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After extracting the protein with RIPA buffer containing protease inhibitor (Roche), the determination of protein concentrations was performed with an enhanced BCA protein assay kit (Beyotime), followed by Western blotting analysis as described previously (54 (link)). Anti-HA (catalog H6908) was from Sigma-Aldrich. Anti-CKS1 (catalog sc-376663) was purchased from Santa Cruz. Anti-SKP2 (catalog A4046), anti-FN1 (catalog A16678), anti-CCNB1 (catalog A19037), and anti-H3 (catalog A22348) Abs were purchased from Abclonal. Anti-FOXM1 (catalog 20459S), anti-ZEB1 (catalog 83243SF), and anti-GAPDH (catalog 2118S) were purchased from Cell Signaling Technology. Anti-FLAG (catalog 20543-1-AP) and anti-RNF112 Ab (catalog 25165-1-AP) were purchased from Proteintech. Anti-tubulin (catalog AT819) was purchased from Beyotime.
9
Western Blot Analysis of Adipogenic Markers
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Protein extractions were prepared with RIPA containing 1mM PMSF (Beyotime Institute of Biotechonogy, Haimen, China). Then the protein extractions were subjected to Western blot analysis, as previously described [38 (link),40 (link)]. Antibodies to FABP4 (Santa Cruz Biotechnology, sc-271529, 1:1000), Antibodies to PPARγ (Santa Cruz Biotechnology, sc-7196, 1:200), Antibodies to C/EBPa (Cell Signaling Technologies, #8178, 1:1000), and Anti-Tubulin (Beyotime Biotechnology, AT819, 1:2000) were used as primary antibodies. After being incubated with HRP-labeled goat anti-mouse IgG (Beyotime Biotechonogy, A0216, 1:1000) or anti-rabit IgG (Beyotime Biotechonogy, A0208, 1:1000), the blots were visualized using electrochemiluminescence (ECL; Millipore, Darmstadt, Germany). The band signal intensities in the western blots were quantified by ImageJ software (https://imagej.nih.gov/ij/ ).
10
Western Blot Analysis of Muscle Proteins
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Briefly, cells were lysed in the radio immune precipitation assay (RIPA) buffer (Beyotime, Shanghai, China) containing phenylmethane sulfonyl fluoride (PMSF) protease inhibitor (Beyotime, Shanghai, China). After incubation on ice for 30 min, the samples were centrifuged at 10,000 g for 10 min at 4 °C, and the supernatant was collected. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes (Whatman, Maidstone, UK), then membranes were probed with primary and secondary antibodies. The primary antibodies used were anti-MYHC (1:1000, B103; DHSB, Iowa City, IA, USA), anti-GAPDH (1:1500, AB-P-R 001, Hangzhou Goodhere Biotech, Hangzhou, China), and anti-Tubulin (1:1000, Beyotime, Shanghai, China). The secondary antibodies used were goat anti-rabbit IgG-HRP (1:5000, BA1054, Boster, Wuhan, China) and peroxidase-goat anti-mouse IgG (1:2500, BA1051, Boster, Wuhan, China). Image J software (d1.47, National Institutes of Health, Bethesda, MD, USA) was used to quantify the band intensity.
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