The slices were deparaffinized in xylene, rehydrated with ethanol, and washed with phosphate-buffered saline (5 min per wash, three times) at room temperature. Hydrogen peroxide (3%) was used to treat the slices for 10 min to block the activity of endogenous peroxidase. Antigen retrieval was performed by microwave treatment for 10 min in 10 mm of sodium citrate (pH 6.0). Then, slices were incubated overnight at 4 °C with anti-JAK1 (Proteintech, Wuhan, China) and anti-STAT3 antibodies (Proteintech, Wuhan, China) at a dilution of 1:50. Negative controls did not include a primary antibody. Then, photographs of five random sections of each slice were taken with the same background. The percentage of JAK1 and STAT3 positive cells to total cells was calculated. ImageJ software was used as the image analysis tool.
Anti jak1
Manufactured by Proteintech
Sourced in United States
Anti-JAK1 is a primary antibody that specifically recognizes the JAK1 protein. JAK1 is a Janus kinase that plays a crucial role in signal transduction pathways associated with cytokine and growth factor receptors.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti stat1, by Proteintech (1 mentions)
Anti gapdh, by Yeasen (1 mentions)
Anti p stat1, by Abcam (1 mentions)
Anti cxcl10, by Abcam (1 mentions)
Anti tublin, by Yeasen (1 mentions)
Rabbit peroxidase conjugated secondary antibody, by ABclonal (1 mentions)
Anti pd l1, by Proteintech (1 mentions)
Anti stat3, by Proteintech (1 mentions)
Anti pstat3, by Affinity Biosciences (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Bca protein quantification kit, by Biosharp (1 mentions)
Anti stat1, by Cell Signaling Technology (1 mentions)
Anti pjak1 tyr1034 1035, by Cell Signaling Technology (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Anti flag, by Cell Signaling Technology (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Human fibronectin, by Thermo Fisher Scientific (1 mentions)
Anti stat1, by Abcam (1 mentions)
5 protocols using anti jak1
1
Immunohistochemical Analysis of JAK1 and STAT3
2
Western Blot Profiling of HPV Biomarkers
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Cultured cells were lysed with lysis buffer. Equal amounts of protein were run on 10% SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (Millipore). After blocking with 5% milk in TBST, membranes were incubated with primary antibodies overnight. The following antibodies were used: anti-HPVE6 (1:1000, Arigo), anti-HPVE7(1:1000, Bioss), (anti-CXCL10 (1:1,000, Abcam), anti-CXCR3 (1:1,000, Boster), anti-PDL1 (1:2,000, proteintech), anti-STAT1 (1:1,000, proteintech), anti-pSTAT1 (1:1,000, Abcam), anti-JAK1 (1:2,000, proteintech), anti-GAPDH (1:6,000, Yeasen) and anti-tublin (1:3,000, Yeasen). Membranes were then incubated with the rabbit peroxidase-conjugated secondary antibody (1:10,000, Abclonal). The blots were detected by sensitive chemiluminescence liquid analysis (Yeasen) and Biorad software was used to capture the images.
3
Western Blot Analysis of JAK/STAT Pathway
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We extracted total protein from cells using RIPA lysis buffer. Protein quantification was performed using BCA Protein Quantification Kit (Biosharp, Hefei, China) according to the protocol. Sample protein (25 μg) were loaded, followed by SDS-PAGE electrophoresis. Then the purpose tape was transferred to polyvinylidene fluoride (PVDF) membranes. After rinsed with TBST for 5 min (3 times), the membranes were blocked with 5% skim milk for 2 h at 37°C. After washing by TBST for three time, membranes were immersed in the primary antibodies incubation solution (anti-JAK1; dilution 1:1000; Proteintech, Chicago, IL, USA); anti-STAT3 (dilution 1:1000; Proteintech); anti-p-STAT3 (dilution 1:1000; Affinity, Jiangsu, China); anti-GAPDH (dilution 1:5000; Proteintech) overnight at 4°C. The secondary antibodies (goat anti-rabbit or goat anti-mouse HRP-conjugated IgG; dilution 1:5000; ZSGB-BIO, Beijing, China) were added with the membranes at 37°C for 1 h. ECL luminescent solution was added to the membrane, and images were taken using Integrated chemiluminescence instrument. Quantitative analysis was performed using Image J software.
4
Murine Cancer Cell Lines and Immunoblotting
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The mouse cancer cell lines B16 and LLC were purchased from ATCC (Manassas, VA). B16-OVA and MC38 cells were generously provided by Dr. Pan Zheng (University of Maryland; Baltimore, MD). All cell lines are mycoplasma free. The cells were maintained in Dulbecco’s modified Eagle’s medium or Roswell Park Memorial Institute 1640 medium (RPMI-1640) supplemented with 10% FBS, 1% penicillin-streptomycin, 2mM L-glutamine, and 1 mM sodium pyruvate at 37 °C in 5% CO2.
For western blot, the anti-CNR2 antibody was purchased from Invitrogen (CA, USA). Anti-pJAK1-Tyr1034/1035, anti-pSTAT1-Ser727 antibodies, anti-STAT1, anti-pSTAT3-Ser727 antibodies, anti-STAT3, anti-Flag, and secondary antibodies HRP-mouse or HRP-rabbit were purchased from Cell Signaling Technology (Boston, Massachusetts USA). Anti-β-actin, anti-JAK1 were purchased from Proteintech (Chicago, IL, USA).
AEA and THC were purchased from Sigma (Sigma‐Aldrich, CA, USA). The anti-mouse PD-1 antibody was purified from hybridoma (clone G4; kindly provided by Dr. Lieping Chen, Yale University, New Haven, CT) culture supernatant.
For western blot, the anti-CNR2 antibody was purchased from Invitrogen (CA, USA). Anti-pJAK1-Tyr1034/1035, anti-pSTAT1-Ser727 antibodies, anti-STAT1, anti-pSTAT3-Ser727 antibodies, anti-STAT3, anti-Flag, and secondary antibodies HRP-mouse or HRP-rabbit were purchased from Cell Signaling Technology (Boston, Massachusetts USA). Anti-β-actin, anti-JAK1 were purchased from Proteintech (Chicago, IL, USA).
AEA and THC were purchased from Sigma (Sigma‐Aldrich, CA, USA). The anti-mouse PD-1 antibody was purified from hybridoma (clone G4; kindly provided by Dr. Lieping Chen, Yale University, New Haven, CT) culture supernatant.
5
Molecular Assays for Antiviral Signaling
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LB broth (catalog# ST156, Shanghai, China), BeyoECL Plus (catalog# P0018S), Bradford protein assay kit (catalog# P0006), BeyoFast™ SYBR Green qPCR Mix (2X, catalog# D7260), penicillin–streptomycin (catalog# C0222), fetal bovine serum (FBS, catalog# C0225), Beyozol (catalog# R0011), BeyoRT™ II cDNA synthesis reagent (catalog# D7168M), RIPA lysis buffer (catalog# P0013C), and bovine serum albumin (BSA, catalog# ST025-5 g) were purchased from Beyotime Biotechnology. Phosphate-buffered saline (PBS) was purchased from Thermo Fisher Scientific (pH 7.4, catalog# 10010072). Dulbecco's modified Eagle’s medium (DMEM) was purchased from Thermo Fisher Scientific (catalog# 11995073). BEGM media with SingleQuot kit additives were purchased from Lonza (catalog# CC-3170, Walkersville, MD). Human fibronectin (catalog# PHE0023) was purchased from Thermo Fisher Scientific (catalog# PHE0023, Waltham, MA). Bovine collagen type I was purchased from STEMCELL (catalog# 07001, STEMCELL). Anti-IFN-α polyclonal antibody (catalog# 66162-1-Ig, Proteintech), Anti-IFN-β polyclonal antibody (catalog# 27506-1-AP, Proteintech), Anti-JAK1 (rabbit monoclonal, catalog# ab133666), anti-STAT1 (rabbit monoclonal, catalog# ab234400), anti-GAPDH (rabbit monoclonal, catalog# ab8245), and anti-STAT2 (rabbit monoclonal, catalog# ab32367) were purchased from Abcam.
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