Immunoblotting was carried out as described previously [11 (link)]. The following antibodies were purchased from Abcam: anti-GAPDH (1:2500, 37 kDa, AB9485, RRID:AB_307275), anti-IL-6 (1:2000, 17 and 50 kDa, ab9324, RRID:AB_307175), and anti-ICAM-1 (1:4000, 90 kDa, AB53013, RRID:AB_870702). Anti-ACE2 (1:1000, 125 kDa, MA532307, RRID:AB_2809589) and anti-HIF-1α (1:1000, 130 kDa, PA5-85,494, RRID:AB_2792634) antibodies were obtained from Thermo Scientific. The following antibodies were purchased from Cell Signaling Technology: anti-ERK1/2 (1:1000, 42, 44 kDa, #4695, RRID:AB_390779), anti-phospho-ERK1/2 (Thr202/Tyr204), (1:1000, 42, 44 kDa, # 9101, RRID:AB_331646), anti-MSK1 (1:1000, 95 kDa, #3489, RRID:AB_2285349), anti-phospho-MSK1 (Thr581) (1:1000, 95 kDa, #9595, RRID:AB_2181783), anti-p38 (1:1000, 43 kDa, #9212, RRID:AB_330713), anti-phospho-p38 (Thr180/Tyr182) (1:1000, 43 kDa, #4511, RRID:AB_2139682), anti-phospho-JNK (Thr183/Tyr185) (1:1000, 46, 54 kDa, #4668, RRID:AB_823588), anti-JNK (1:1000, 46, 64 kDa, #9252, RRID:AB_2250373), anti-phospho-NFκB p65 (Ser536) (1:1000, 70 kDa, #3033, RRID:AB_331284), and anti-NFκB p65. (1:1000, 70 kDa, #6956, RRID:AB_10828935). The following antibody were obtained from ABclonal (Woburn, MA): anti-phospho-NFkB p65 (S276) (1:1000, 70 kDa, AP0123, RRID:AB_2771505). An anti-phospho-MBP antibody (1:200, 18 kDa, 13–104) was purchased from Merck.
Anti hif 1α
Manufactured by Thermo Fisher Scientific
Sourced in France
Anti-HIF-1α is a laboratory reagent used for the detection and quantification of HIF-1α (Hypoxia-Inducible Factor-1 alpha) in various biological samples. It is a crucial tool for researchers studying cellular responses to hypoxia and related molecular pathways.
Automatically generated - may contain errors
Lab products found in correlation
Ab53013, by Abcam (1 mentions)
Ab9324, by Abcam (1 mentions)
Anti icam 1, by Abcam (1 mentions)
Anti ace2, by Thermo Fisher Scientific (1 mentions)
Anti msk1, by Cell Signaling Technology (1 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (1 mentions)
Anti il 6, by Abcam (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti jnk, by Cell Signaling Technology (1 mentions)
Ab9485, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti mdr1, by Proteintech (1 mentions)
Horseradish peroxidase hrp conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti mouse igg, by Cell Signaling Technology (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Enhanced chemiluminescence detection kit, by Cytiva (1 mentions)
Anti p62, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
7 protocols using anti hif 1α
1
Immunoblotting Antibody Validation Protocol
2
Protein Analysis in Cancer Cell Lines
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Protein samples from patient tissues and SMMC-7721 cells were prepared using radio-immunoprecipitation assay lysis buffer (RIPA; Beyotime Institute of Biotechnology, China), and the protein concentration was detected by the BCA protein quantitation kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Western blot analysis was then performed based on protocols reported in a previous study (22 (link)). The following primary and secondary antibodies were used: Anti-VEGF (cat. no. 19003-1-AP; 1:2,000, ProteinTech, Inc., Chicago, IL, USA), anti-MDR1 (cat. no. PA5-28810; 1:2,000, Thermo Fisher Scientific, Inc.), anti-HIF-1α (cat. no. 20960-1-AP; 1:2,000, ProteinTech, Inc.), anti-β-actin (cat. no. MA1-140; 1:2,000, Thermo Fisher Scientific, Inc.), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (cat. no. 7074; 1:2,000, Cell Signaling Technology, Inc., Danvers, MA, USA) and HRP-conjugated anti-mouse IgG (cat. no. 7076; 1:2,000; Cell Signaling Technology, Inc.) antibodies.
3
Western Blot Analysis of Autophagy Markers
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Cells were lysed in radioimmune precipitation assay buffer (150 mM NaCl, 0.5% sodium deoxycholate, 50 mM Tris-HCl, pH 8, 0.1% SDS, 0.1% Nonidet P-40) supplemented with protease inhibitors (Roche, Boulogne-Billancourt, France). Proteins were separated on SDS/PAGE gels, transferred to nitrocellulose membranes (Amersham Biosciences, Velizy-Villacoublay, France), and blocked with 5% non-fat milk in PBS containing 0.1% Tween-20. Membranes were then incubated overnight at 4 °C with the relevant primary antibodies: anti-HIF-1α (Thermo Scientific, Illkirch, France), anti-LC3 (Sigma-Aldrich, Saint-Quentin Fallavier, France), anti-p62 (Cell Signaling Technology, Saint-Cyr-L’École, France), and anti-β-actin (Cell Signaling Technology, Saint-Cyr-L’École, France). After washes, membranes were incubated with the appropriate HRP-conjugated secondary antibodies (Cell Signaling Technology, Saint-Cyr-L’École, France). Blots were detected using the Enhanced Chemiluminescence Detection kit (Amersham Biosciences, Velizy-Villacoublay, France), revealed using the ChemiDocTM XRS System (Bio-Rad, Marnes-la-Coquette, France), and quantified using the Image Lab Software (Bio-Rad, Marnes-la-Coquette, France).
4
Poly-G3 and Poly-T3 Impact on Treg Cells
5
Immunohistochemical Analysis of Tumor Markers
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Deparaffinized 5‐ to 6‐μm sections of formalin‐fixed paraffin‐embedded (FFPE) mouse xenograft tumor and human colorectal carcinoma tissues followed by pressure cooker antigen retrieval in citrate buffer were blocked in 3% H2O2 and goat or horse serum plus MOM block reagent (if mouse primary antibody was used on mouse tissue), avidin, and biotin blocking reagents (Vector Labs). Sections were incubated in primary antibodies: antiphospho‐PDHpSer293 (Calbiochem; 1:50–1:100), anti‐β‐catenin (BD; 1:500), anti‐LEF‐1 (Cell Signaling; 1:100), anti‐HIF1α (Thermo Scientific; 1:1,000) followed by biotinylated secondary antibodies and visualization using a peroxidase‐conjugated avidin‐based Vectastain protocol. Slides were then counterstained with hematoxylin and mounted using Permount mounting medium (Fisher). Images were captured using an Olympus FSX100 system and processed in Adobe Photoshop.
6
Western Blot Analysis of Larval Proteins
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Total protein extracts were obtained by homogenization of pools of 20 6-dpf larvae in ice cold RIPA buffer (ThermoFisher, 89900) and Complete EDTA-free protease inhibitor cocktail (Sigma, 11873580001). For western blot analysis 40 μg of protein extracts were loaded per well on Bolt 4–12% Bis-Tris Plus Gels (ThermoFisher, NW04120BOX) and blotted on PVDF immobilon-p membranes (Millipore, IPFL00010). Dried membranes were then washed with PBS (Sigma, P4417) with 0.1% (w/v) Tween20 and incubated overnight with primary antibodies at 4 °C: anti-HIF1α (1:500, MA1-16504 Invitrogen); anti-STAT3 (1:1000, 9139 S Cell Singaling); anti-pSTAT3 Y705 (1:1000, D3A7 Cell Signaling), anti-pSTAT3 S727 (1:1000, 9134 Cell Signaling) and anti-βActin (1:5000, MA1-744 ThermoFisher). Secondary anti-Rabbit and anti-Mouse HRP-conjugated antibodies (1:5000, 170-6515 BIORAD, and 1:5000, 170-6516 BIORAD, respectively) were incubated for 1 h at room temperature and protein bands detected by chemiluminescence on an Alliance MINI HD 9 Blot Imaging System. Quantification of the signal was performed with ImageJ.
7
Immunohistochemistry Panel for Cancer Markers
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Immunohistochemistry was performed on standard tissue sections using an automated IHC stainer (Ventana, Tucson, AZ). In this study, we included 18 IHC markers. The primary antibodies used for the detection of these 18 proteins were as follows: anti-PAX-8 (Clone MRQ-50, Ventana), anti-P504S (Clone SP116, Ventana), anti-Ki-67 (Clone 30-9, Ventana), anti-CD10 (Clone SP67, Ventana), anti-HER2 (Clone 4B5, Ventana), anti-PTEN (Clone SP218, Ventana), anti-COX2 (Clone SP21, Ventana), anti-Vimentin (Clone Vim 3B4, Ventana), anti-TFE3 (Clone MRQ-37, Ventana), anti-CA9 (Cat No. 5649, Cell Signaling Technology), anti-CD117 (Cat No. 37805, Cell Signaling Technology), anti-mTOR (Cat No. 2983, Cell Signaling Technology), anti-CK7 (Cat No. ab181598, Abcam), anti-BAP1 (Cat No. ab199396, Abcam), anti-HGF (Cat No. ab83760, Abcam), anti-SETD2 (Cat No. PA5-43071, Invitrogen), anti-HIF-1α (Cat No. MA1-516, Invitrogen) and anti-PBRM1 (Cat No. HPA015629, Sigma-Aldrich). Criteria for the positive and negative scoring of IHC specimens for a given protein marker has been described previously (24 (link)), and all samples were evaluated by two independent experienced pathologists.
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