Total proteins were extracted by RIPA buffer supplemented with PMSF (Solarbio, Beijing, China) and quantified by a BCA kit. Then, the proteins were separated in SDS-PAGE gels and transferred into PVDF membranes (Millipore, Massachusetts, USA). Membranes were blocked with TBST with 5% skim milk powder and incubated overnight at 4 °C with primary antibodies against YAP1 (1:1000), P-YAP1 (1:1000), VEGFA (1:1000), Hexokinase II (HK2; 1:5000), LDHA (1:2000), HIF1A (1:1000), GAPDH (1:5000), Tublin (1:1000), and β-actin (1:1000) (Proteintech, Wuhan, China). The next day, blots were washed with PBS and then secondary antibodies were incubated with the membrane at room temperature for 1 h. The membrane was visualized using a chemiluminescence kit (Absin, Shanghai, China) and quantified by densitometry analysis using ImageJ software. GAPDH and tubulin were used as loading controls.
Hif1a
Manufactured by Proteintech
Sourced in China, United States
HIF1A is a protein that functions as a transcription factor, playing a crucial role in the cellular response to reduced oxygen levels (hypoxia). It regulates the expression of genes involved in angiogenesis, glucose metabolism, and cell survival under hypoxic conditions.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Proteintech (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Vegfa, by Proteintech (2 mentions)
Gapdh, by Proteintech (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Proteintech (2 mentions)
Bcl 2, by Proteintech (2 mentions)
Snail, by Proteintech (2 mentions)
Chemiluminescence kit, by Absin (1 mentions)
P yap1, by Proteintech (1 mentions)
Tublin, by Proteintech (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Glut1, by Proteintech (1 mentions)
Uqcrc2, by Proteintech (1 mentions)
Mtco2, by Proteintech (1 mentions)
Bicinchoninic acid (bca), by Selleck Chemicals (1 mentions)
Atp5a, by Proteintech (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Gapdh, by Abcam (1 mentions)
8 protocols using hif1a
1
Protein Expression Analysis in Cell Metabolism
2
Molecular Mechanisms in Cell Metabolism
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BCA was purchased from Selleck Chemicals (Shanghai, China). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Kumamoto, Japan). PE Annexin V apoptosis detection commercial kit was purchased from BD biosciences (Shanghai, China). Reactive Oxygen Species (ROS) Assay Kit was purchased from Beyotime Biotechnology (Shanghai, China). Cell-Light EdU Apollo567 In Vitro Kit was purchased from Guangzhou Ruibo Biotechnology Co. LTD (Guangzhou, China). Bax, Bcl-2, ND1, SHDB, UQCRC2, MTCO2, ATP5A, MFN2, GLUT1, HK2, PMK2, LDH, HIF-1a, and Drp1 antibodies were obtained from proteintech (Wuhan, China). AKT, p-AKT, mTOR, and p-mTOR antibodies were obtained from Cell Signaling Technology. LC3B, Beclin-1, P62, and GAPDH were obtained from ABCAm (Cambridge, UK).
3
Protein Expression Analysis by Western Blot
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Cell proteins were extracted using dissolved buffers. Boil the protein in a metal bath for 10 min to denature it. Protein samples were isolated using SDS-PAGE (Beyotime Biotechnology) and then transferred to PVDF membrane (Millipore). Soak in 5 % skim milk for 1 h. Refrigerate the film at 4 °C overnight with the primary antibody (diluted to a concentration of 1:2000), followed by incubation at room temperature for 1 h with the secondary antibody (diluted to a concentration of 1:5000). The strips were exposed to ECL luminescent solution (Thermo). Antibodies to HIF-1A, E-cadherin, Vimentin, Snail, β-actin, ITGB1 were purchased from Proteintech (China).
4
Western Blot Analysis of Protein Expression
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The cells were harvested in radioimmunoprecipitation assay buffer and lysed on ice. From each sample, equal amounts of protein were employed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes. After being blocked with 5% nonfat milk/TBST, the membrane was successively incubated with the appropriate primary antibody and a secondary antibody. Every incubation was followed by 3 washes with TBST. Protein signals were determined using a chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA). Primary antibodies: MSH6, N-cadherin, Vimentin, E-cadherin, p-AKT, p-STAT3, p-Smad2/3, p-ERK, p-p38, p-JNK, p-p65, STAT3, Smad2/3, ERK, p38, JNK and p65 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA); Ki67, Nestin, CD133, SOX2, Cyclin D1, Bax, Bcl-2, MMP2, MMP9, HIF1A, VEGFA, Snail, Slug, Twist, ZEB1, ZEB2, AKT, TGFB1 and β-actin antibodies were supplied by Proteintech (Wuhan, Hubei, China); CXCR4 antibody was obtained from Abcam (Shanghai, China). Secondary antibodies: horseradish peroxidase (HRP)-conjugated affinipure goat anti-mouse IgG and HRP- conjugated affinipure goat anti-rabbit IgG were purchased from Proteintech (Wuhan, Hubei, China).
5
Comparative Protein Expression Analysis of Colorectal Cells
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The total proteins of SW480 and NCM460 cells were prepared using RIPA buffer containing protease and phosphatase inhibitors. A BCA protein assay kit was used to measure protein concentrations. 20 µg proteins were loaded per lane, separated by electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes (C3117, Millipore). The membrane was blocked and then incubated for 1 h with β-actin (1:100000; ABclonal, AC026), PPAR gamma (1:5000; Proteintech, 16643-1-AP), MMP9 (1:1000; Proteintech, 10375-2-AP), TNF alpha (1:3000; Proteintech, 60291-1-IG), TGF beta1 (1:2000; Proteintech, 21898-1-AP), COX2 (1: 500; Proteintech, 27308-1-AP), HIF1A (1:2500; Proteintech, 20960-1-AP) and Beta-catenin (1:10000; Proteintech, 51067-2-AP). Immunoblot analysis was performed with horseradish peroxidase (HRP)-conjugated anti-mouse antibodies or anti-rabbit antibodies (1:5000; ZSGB-BIO, ZB-5301, and ZB-5305) and developed with the ECL kit (Beyotime Biotechnology, P0018FM). The level of β-actin was used as a loading control, and the ratios of the gray value of the target protein bands to the gray value of the corresponding internal control bands were defined as the expression level of the target protein.
6
Protein extraction and Western blot analysis
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Total protein was isolated using RIPA buffer (Applygen, Beijing, China) with protease inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). Nuclear and cytoplasmic protein was isolated using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Immunoblotting was performed with primary antibodies against ANXA2 (1:200), p-ANXA2 (Tyr23, 1:200), VEGF (1:200) (Santa Cruz Biotechnology, Dallas, Texas, USA), MYC (1:1000), p-SRC (Tyr418, 1:1000) (Abcam, Cambridge, UK), Ubiquitin (1:500), Histone H3 (1:1000) (CST, Danvers, MA, USA), HIF1A (1:500), SRC (1:500), HA-tag (1:3000), His-tag (1:3000) (Proteintech, Wuhan, China). GAPDH (1:500) (Proteintech) was used as a loading control. Secondary antibodies (Goat anti-Mouse IgG and Goat anti-Rabbit IgG, 1:5000) were purchased from Applygen. The signals were visualized with a super enhanced chemiluminescence (ECL) detection reagent (Applygen). Quantitative analysis of immunoblotting was performed using ImageJ (Ver. 1.52a, NIH image, Bethesda, MD, USA).
7
Protein Extraction and Western Blotting Analysis
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Total proteins were extracted from model mouse lung tissue and cells using a prepared SDS lysis buffer for Western blotting analysis. Then, 60 ug of protein was isolated using 12% SDS-polyacrylamide gel and imprinted onto polyvinylidene fluoride membrane (PVDF, Merck Millipore, Germany). Then, the membrane was sealed in 5% skim milk for 1.5 h. β-actin (Abcam, # ab8226), Collagen I (Arigo Biolaboratories, #ARG21965), HIF-1a (Proteintech, #20960-1-AP), LC3A/B (Cell signaling technology, #12741), P62 (Cell signaling technology, #16177S), Phospho-SQSTM1/p62(Ser349) (CST, #E7M1A), Bcl-2 (Abcam, #ab182858), BNIP3 (Santa Cruz Biotechnology, #sc-56167), BNIP3L (Proteintech, #12986-1-AP), BECN1 (Boster, #PB0014), and SOD2 (Boster, #BA4566) were used. The PVDF membranes were subjected to incubation with antibodies above at 4 °C overnight, then HRP-labeled secondary antibody (ZSGB-BIO, Beijing, China) was added and incubated for 1 h, and the immune reaction was detected by the ECL luminescence system.
8
Quantitative Western Blot Analysis
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Western blotting was performed as described previously (Yu et al., 2019 (link)). In brief, protein samples were separated in 10–15% SDS-PAGE acrylamide gels, transferred onto PVDF membranes (Millipore, 0.22μm and blocked with 5% skim milk for 2h). The blots were then incubated at 4°C overnight with the following primary antibodies: Claudin-5 (1:1,000; Invitrogen, 34–1,600), LC3 (1:1,000; ProteinTech, 14600-1-AP), Caveolin-1 (1:200; Santa Cruz Biotechnology, sc-894), HIF-1a (1:1000; ProteinTech, 20960-1-AP), iNOS (1:1,000; ProteinTech, 18985-1-AP), LAMP-1 (1:1000; ProteinTech, 21997-1-AP), ATP1A1 (1:1,000, ProteinTech, 14418-1-AP), or β-actin (1:1,000; Servicebio, GB12001). Then, the blots were incubated at room temperature for 2h with HRP conjugated anti-rabbit or anti-mouse IgG (H+L; 1:1,000; Servicebio) secondary antibodies. Western blot bands were analyzed by adding ECL advance Western blotting detection reagents (Thermo, 34,580) and imaged using a SmartChemi-500 imaging system (Sage Creation Science, China). The immunoblot bands were quantitatively analyzed by ImageJ software.
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