Hypoxia-treated cells were rapidly lysed with 1% SDS and boiled in SDS loading buffer and analyzed by Western blot assays. The following antibodies were used: anti-ALKBH5 (1:1000, Proteintech, 16837–1-AP), anti-HIF1a (1:1000, GeneTex, GTX127309), anti-FTO (1:1000, Abclonal A3861), anti-METTL3 (1:1000, Abclonal, A19079), anti-b-Tubulin (1:10,000, Abclonal, AC021), anti-SFPQ (1:1000, Abclonal, A3494), anti-NONO (1:1000, Abclonal, A3800), anti-PSPC1 (1:1000, Proteintech, 116714-1-AP), and anti-GFP (1:1000, Proteintech, 50430-2-AP). Direct-load Color Prestained Protein Marker (GenStar, M221) was used in all assays.
Anti β tubulin
Manufactured by ABclonal
Sourced in China, United Kingdom
Anti-β-tubulin is a laboratory reagent used in research applications. It is an antibody that specifically binds to the β-tubulin protein, a key component of the cytoskeleton in eukaryotic cells. This antibody can be used to detect and analyze the distribution and expression levels of β-tubulin in cells and tissues through various experimental techniques, such as Western blotting, immunofluorescence, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by ABclonal (3 mentions)
Ab77262, by ABclonal (2 mentions)
S protein agarose beads, by Merck Group (2 mentions)
Anti vhl, by ABclonal (2 mentions)
Anti epas 1 hif 2 alpha, by Santa Cruz Biotechnology (2 mentions)
Anti hdac1, by ABclonal (2 mentions)
Anti gapdh, by Transgene (2 mentions)
Anti ha, by Santa Cruz Biotechnology (2 mentions)
Anti flag, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (2 mentions)
Anti gfp, by Proteintech (1 mentions)
Anti mettl3, by ABclonal (1 mentions)
Anti alkbh5, by Proteintech (1 mentions)
Anti fto, by ABclonal (1 mentions)
Anti sfpq, by ABclonal (1 mentions)
Gtx114209, by ABclonal (1 mentions)
Direct load color prestained protein marker m221, by GenStar (1 mentions)
Anti caspase 3, by Proteintech (1 mentions)
Anti gapdh, by OriGene (1 mentions)
9 protocols using anti β tubulin
1
Hypoxia-induced Protein Expression Analysis
2
Whole-Cell Protein Extraction and Analysis
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To obtain whole-cell protein extracts, cells were harvested and lyzed as previously described (22) . Cell culture medium and serum from the allografted mice were prepared to measure IL8 levels. The corresponding primary antibodies used for immunoblotting included anti-ALKBH5 (1:1,000, Proteintech, 16837-1-AP), anti-HIF1a (1:1,000, GeneTex, GTX127309), anti-FTO (1:1,000, Santa Cruz Biotechnology, sc-271713), anti-METTL3 (1:1,000, Abclonal, A19079), anti-b-actin (1:10,000, Abclonal, AC026), anti-b-tubulin (1:10,000, Abclonal, AC021), anti-GAPDH (1:20,000, Abclonal, AC002), anti-IL8 (1:1,000, GeneTex, GTX115959), anti-albumin (1:1,000, Abclonal, A0353), anti-SFPQ (1:1,000, GeneTex, GTX114209), and anti-NONO (1:1,000, Abclonal, A3800). And Direct-load Color Prestained Protein Marker (M221, GenStar) was used in all Western blot assays.
3
Comprehensive Protein Expression Analysis
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Western blot assays were performed as previously described [29 (link)]. The primary antibodies used in this study were anti-SOX9 (#A2479, ABclonal, Wuhan, China; 1:2000), anti-PCNA (#13110, Cell Signaling Technology, Shanghai, China; 1:1000), anti-Ki67 (#ab16667, Abcam, Shanghai, China; 1:1000), anti-CDK2 (#ab232753, Abcam, Shanghai, China; 1:1000), anti-CYP11A1 (#D122183, Sangon, Shanghai, China; 1:1000), anti-CYP19A1 (#A2161, ABclonal, Wuhan, China; 1:1000), anti-StAR (#8449S, Cell Signaling Technology, Shanghai, China; 1:1000), anti-caspase3 (#19677-1-AP, Proteintech, Wuhan, China; 1:1000), anti-GAPDH (#TA802519, ORIGENE, Wuxi, China; 1:3000), and anti-β-tubulin (#AC008, ABclonal, Wuhan, China; 1:3000). The corresponding HRP-conjugated secondary antibodies obtained from Sangon Biotech (Shanghai, China) were diluted in 0.25% BSA/TBST solution. The protein levels of β-tubulin and GAPDH served as internal controls in oxidative and non-oxidative experiments, respectively. Each group had three independent biological replicates. The replicates with the highest representativeness were selected and are shown in the figures, and their corresponding normalized fold change values were calculated and are listed under the blot images.
4
Co-immunoprecipitation and Subcellular Fractionation Protocol
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Cells were transfected with appropriate plasmids and lysed by co-immunoprecipitation lysis buffer (10% glycerol, 0.5% NP-40, 150 mM NaCl, 0.1 mM EDTA) supplemented with protease inhibitor cocktail (Roche). Cell lysates were incubated with the S-protein Agarose beads (Millipore, 69704) or indicated primary antibody and protein A/G agarose beads (Santa Cruz Biotechnology, sc-2003). The immunocomplexes were then washed by PBSN (PBS containing 0.1% NP-40) three times and subjected to SDS-Page. For subcellular fractionation, nuclear and cytoplasmic extracts were isolated with a nuclear-cytoplasmic extraction kit (Applygen, P1200) following the manufacturer’s protocol.
Antibodies used in this study were as follows: anti-VHL (Abcam, ab77262) for Western Blot, anti-VHL (Abclonal, A0377) for Immunohistochemistry, anti- EPAS-1/HIF-2 alpha (Santa Cruz Biotechnology, sc-13596), anti-BICD2 (Abcam, ab237616), anti-GAPDH (TransGen Biotech, HC301-01), anti-β-Tubulin (ABclonal, AC021), anti-β-Actin (ABclonal, AC004), anti-HDAC1 (ABclonal, A19571), anti-FLAG (Sigma, F3165), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-GFP (MBL, 598).
Antibodies used in this study were as follows: anti-VHL (Abcam, ab77262) for Western Blot, anti-VHL (Abclonal, A0377) for Immunohistochemistry, anti- EPAS-1/HIF-2 alpha (Santa Cruz Biotechnology, sc-13596), anti-BICD2 (Abcam, ab237616), anti-GAPDH (TransGen Biotech, HC301-01), anti-β-Tubulin (ABclonal, AC021), anti-β-Actin (ABclonal, AC004), anti-HDAC1 (ABclonal, A19571), anti-FLAG (Sigma, F3165), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-GFP (MBL, 598).
5
RNA Extraction and Real-Time PCR Analysis
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Total RNA was extracted using the FastPure total RNA isolation kit (Vazyme Biotech Co., Nanjing, China) and reverse-transcribed to complementary DNA using HiScript III All-in-one RT SuperMix (Vazyme Biotech Co, Nanjing, China), according to the manufacturer’s instructions. Real-time PCR was performed with SYBER Green PCR Master Mix (Vazyme Biotech Co, Nanjing, China) using a StepOnePlus PCR system (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal normalizer. Primer sequences are noted in the Supplementary materials . Western blot was used to measure α-SMA expression levels in CFs. The primary antibodies were anti-α-SMA (Abcam, Cambridge, UK) and anti–β-tubulin (ABclonal, Wuhan, China).
6
Co-immunoprecipitation and Subcellular Fractionation Protocol
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Cells were transfected with appropriate plasmids and lysed by co-immunoprecipitation lysis buffer (10% glycerol, 0.5% NP-40, 150 mM NaCl, 0.1 mM EDTA) supplemented with protease inhibitor cocktail (Roche). Cell lysates were incubated with the S-protein Agarose beads (Millipore, 69704) or indicated primary antibody and protein A/G agarose beads (Santa Cruz Biotechnology, sc-2003). The immunocomplexes were then washed by PBSN (PBS containing 0.1% NP-40) three times and subjected to SDS-Page. For subcellular fractionation, nuclear and cytoplasmic extracts were isolated with a nuclear-cytoplasmic extraction kit (Applygen, P1200) following the manufacturer’s protocol.
Antibodies used in this study were as follows: anti-VHL (Abcam, ab77262) for Western Blot, anti-VHL (Abclonal, A0377) for Immunohistochemistry, anti- EPAS-1/HIF-2 alpha (Santa Cruz Biotechnology, sc-13596), anti-BICD2 (Abcam, ab237616), anti-GAPDH (TransGen Biotech, HC301-01), anti-β-Tubulin (ABclonal, AC021), anti-β-Actin (ABclonal, AC004), anti-HDAC1 (ABclonal, A19571), anti-FLAG (Sigma, F3165), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-GFP (MBL, 598).
Antibodies used in this study were as follows: anti-VHL (Abcam, ab77262) for Western Blot, anti-VHL (Abclonal, A0377) for Immunohistochemistry, anti- EPAS-1/HIF-2 alpha (Santa Cruz Biotechnology, sc-13596), anti-BICD2 (Abcam, ab237616), anti-GAPDH (TransGen Biotech, HC301-01), anti-β-Tubulin (ABclonal, AC021), anti-β-Actin (ABclonal, AC004), anti-HDAC1 (ABclonal, A19571), anti-FLAG (Sigma, F3165), anti-HA (Santa Cruz Biotechnology, sc-7392) and anti-GFP (MBL, 598).
7
Western Blot Analysis of GFPT2 Protein
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The RIPA buffer (Beyotime Biotechnology, Shanghai, PR China) was added to the tissue preserved at 80 °C and crushed using a homogenizer. Cell clusters (GES, AGS, BGC‐823, HGC‐27, and MKN‐28) were collected and added to RIPA buffer (Beyotime Biotechnology). The mixture was then stood on ice for 20 min, centrifuged at 13,000 rpm for 30 min, and the supernatant was collected. Then the protein concentration was determined by BCA kit (Beyotime Biotechnology). A total of 30 μg protein was separated by 12% SDS‐PAGE gel and transferred to 0.45 μm PVDF membrane, sealed for 5 min using Western Quick Block Kit (GenScript, Piscataway, NJ, USA) at room temperature, and the first antibody was added (anti‐GFPT2, 1:1,000, DF15367, Affinity, Jiangsu, PR China; anti‐β‐Tubulin, 1:5,000, A122891, ABclonal, Wuhan, PR China) and incubated overnight at 4 °C. On the second day, the membrane was incubated with the corresponding horseradish peroxidase‐labeled secondary antibody [anti‐Rabbit IgG (H + L), 1:5,000, SA00001‐2, ProteinTech, Wuhan, PR China] at room temperature for 1 h and then treated with ECL reagents (Meilunbio, Dalian, PR China).
8
Western Blotting of Intestinal Proteins
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Total proteins from tissues and organoids were harvested using RIPA Lysis Buffer (Beyotime) with phenylmethyl sulfonyl fluoride (PMSF) protease inhibitor and phosphatase inhibitor. Samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes, followed by blocking with 5% skimmed milk for 1 h. The membranes were then incubated with primary antibodies and the corresponding secondary antibodies (all 1:1000 dilutions): anti-Lgr5 antibody (Abcam, ab75850), anti-Olfm4 antibody (Cell Signaling, 39141), anti-GAPDH antibody (Abclonal, AC001), anti-GPR41 antibody (Abclonal, A12636), anti-GPR43 antibody (Proteintech, 19952-1-AP), anti-β-tubulin (Abclonal, AC015), anti-Axin2 antibody (Proteintech, 20540-1-AP), anti-β-catenin (Abclonal, A19657), anti-Wnt3 antibody (Abclonal, A9328), and HRP conjugated Goat anti-rabbit IgG (Antgene, 72-8067). The bands were detected using a chemiluminescence imaging system (UVP) with ECL chemiluminescence detection kit (Vazyme). The relative fold change in protein expression was normalized to that of GAPDH or β-tubulin.
9
Protein Extraction and Western Blot Analysis
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Protein extraction were carried out, followed by WB analysis as described previously (Zhang et al. 2014 (link)). Proteins were separated with SDS–polyacrylamide gel electrophoresis (PAGE) as well as transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, USA). The primary antibodies were applied according to the provider’s recommendations: anti-MAX (1:200, CST, USA), anti-IGFBP1 (1:1000, Abcam, UK), anti-β-tubulin (1:5000, Abclonal, China). β-Tubulin was used as a loading control. Immunoblotting analysis was performed with Supersignal West Pico Chemiluminescent substrate (Thermo Fisher, USA).
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