At each time point, 30 larvae from control or metronidazole treatment groups were harvested in RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma, 78830), Protease Inhibitor Cocktail (Promega, G6521), Phosphatase Inhibitor Cocktail 2 (Sigma, P5726), and Phosphatase Inhibitor Cocktail 3 (Sigma, P0044). Western blotting was performed using standard protocols [48 (link)]. Eleven primary antibodies were used in this study: anti-IL-7R (1:1500; Abcam, ab180521), anti-MBP (1:500; Anaspec, #55811, Fermont, CA, USA), anti-Phospho-JAK1 (1:1,000; Cell Signaling Technology, #3331, Danvers, MA, USA), anti-Phospho-JAK3 (1:500; Cell Signaling Technology, #5031), anti-STAT3 (1:500; Santa Cruz Biotechnology, sc-7179, TX, USA), anti-Phospho-STAT3 (1:200; Cell Signaling Technology, #9145), anti-STAT5 (1:500; Santa Cruz Biotechnology, sc-836), anti-Phospho-STAT5 (1:200; Cell Signaling Technology, #9351), anti-BCL2 (1:1000; BD Biosciences, 610538), anti-CASPASE-3/cleaved CASPASE-3 (1:300; Wanleibio, WL02117), and anti-CASPASE-7/cleaved CASPASE-7 (1:500; Wanleibio, WL0181). Anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; 1:3,000; Millipore, MAB374, Billerica, MA, USA) was used as a loading control.
Anti phospho jak1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-JAK1 is a primary antibody that recognizes the Janus Kinase 1 (JAK1) protein when phosphorylated. It is designed for the detection of activated JAK1 in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho stat3, by Cell Signaling Technology (5 mentions)
Anti jak1, by Cell Signaling Technology (4 mentions)
Anti stat3, by Cell Signaling Technology (3 mentions)
Anti stat1, by Cell Signaling Technology (3 mentions)
Anti phospho stat1, by Cell Signaling Technology (3 mentions)
Anti bax, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti phospho p38 mapk, by Cell Signaling Technology (2 mentions)
Anti phospho nf κb p65, by Cell Signaling Technology (2 mentions)
Anti p38 mapk, by Cell Signaling Technology (2 mentions)
Anti cox 2, by Cell Signaling Technology (2 mentions)
Anti bcl 2, by BD (1 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Promega (1 mentions)
Anti stat5, by Santa Cruz Biotechnology (1 mentions)
Anti phospho stat5, by Cell Signaling Technology (1 mentions)
Anti mbp, by AnaSpec (1 mentions)
Anti phospho jak3, by Cell Signaling Technology (1 mentions)
6 protocols using anti phospho jak1
1
Western Blot Analysis of Larval Responses
2
Western Blot Analysis of Signaling Proteins
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Cells were lysed by ice-cold lysis buffer. The concentration of protein was measured using BCA kit (Invitrogen, Carlsbad, CA, USA). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene fluoride (PVDF) membranes. Then the membrane was incubated with diluted primary antibody overnight at 4°C. Primary antibodies are shown as follows: anti-phospho-JAK1 (1:1000, #74,129), anti-JAK1 (1:1000, #3344), anti-phospho-STAT3 (1:1000, #9145), anti-STAT3 (1:1000, #12,640), anti-GAPDH (1:1000, #5174), anti-Bax (1:1000, 14796S) (Cell Signaling Technology); anti-Caspase-3 (1:1000, ab197202), and anti-Bcl-2 (1:1000, ab32124) (Abcam, UK). Followed by three times of washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (anti-rabbit, #7074, Cell Signaling Technology) for 1 h at 37°C. The protein bands were visualized by ECL exposure solution, and quantified by a gel imaging system.
3
Western Blot Antibody Procurement
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Anti-iNOS (PA1-036) was purchased from Invitrogen (Carlsbad, CA, USA). Anti-phospho-p44/42 MAPK (Erk1/2) (#9101), anti-p44/42 MAPK (Erk1/2) (#4695), anti-phospho-SAPK/JNK (#9251), anti-SAPK/JNK (#9252), anti-phospho-p38 MAPK (#9211), anti-p38 MAPK (#9212), anti-phospho-NF-κB p65 (#3033), anti-phospho-IκBα (#2859), anti-IκBα (#4814), anti-phospho-JAK1 (#3331), anti-JAK1 (#3332), anti-phospho-STAT1 (#9167), anti-STAT1 (#9172), anti-phospho-STAT3 (#9145), and anti-STAT3 (#30835) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-NFκB p65 (sc-8008) and anti-β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Carlsbad, CA, USA). HRP-conjugated anti-mouse IgG (A21010) and HRP-conjugated anti-rabbit IgG (A21020) were purchased from Abbkine (Wuhan, China). The information on antibodies is summarized in Supplementary Table S2 .
4
Investigating Immune Signaling Pathways
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The following primary antibodies were used in the current study: anti-RIG-I, anti-NF-κB p65, anti-phospho-NF-κB p65 (Ser536), anti-p38 MAPK, anti-phospho-p38 MAPK (Thr180/Tyr182), anti-STAT1, anti-phospho-STAT1 (Tyr701), anti-STAT2, anti-phospho-STAT2 (Tyr690), anti-JAK1, anti-phospho-JAK1(Tyr1034/1035), anti-phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204), anti-p44/p42 MAPK (ERK1/2), anti-phospho-SAPK/JNK MAPK (Thr183/Tyr185), anti-SAPK/JNK MAPK, anti-COX2, and anti-GAPDH (Cell Signaling Technology). Anti-PARP and anti-caspase3 (active form) antibodies were purchased from Gentex. Recombinant human TNF-α, IFN-β, and IFN-λ1 (IL-29) were purchased from Peprotech.
5
Molecular Mechanisms of Ovarian Cancer
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Human ovarian cancer cell lines (SKOV-3, 2774 and OVCAR-3) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Human IFN-α and IFN-γ were obtained from Sigma (St. Louis, MO). The chemical doxazosin was also purchased from Sigma. The JAK1/2 kinase inhibitor INCB18424 (Ruxolitinib) and STAT1 inhibitor (NSC118218) were obtained from Selleck Chemicals (Houston, TX), and stock solutions were prepared in DMSO. NSC74859 (S31-201), a specific STAT3 inhibitor, was purchased from Calbiochem Chemicals (La Jolla, CA). The following primary antibodies were used in this study: anti-JAK1, anti-phospho-JAK1, anti-JAK2, anti-phospho-JAK2, anti-STAT1, anti-phospho-STAT1, anti-STAT3, anti-phospho-STAT3, anti-caspase-3, anti-cMyc, anti-Bcl-2, anti-Bax, anti-p53, anti-survivin, and anti-COX-2 (Cell Signaling, Beverly, MA), anti-PARP, anti-XIAP (BD Biosciences, San Jose, CA), anti-cyclin D1, anti-CDK4, anti-Akt, anti-phospho-Akt, anti-TYK2, anti-phospho-TYK2, anti-PI3K, anti-phospho-PI3K, anti-mTOR, anti-phospho-mTOR, anti-PKCδ, anti-phospho-PKCδ, anti-STAT2, anti-phospho-STAT2, anti-p70S6K, and anti-phospho-p70S6K (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p21, and anti-p27 (Oncogene, San Diego, CA).
6
Interleukin-21 Signaling Pathway Analysis
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Human recombinant IL-21 was purchased from PeproTech EC. The antibodies used were anti-phospho-ERK1/2 (Thr 202/Tyr 204), anti-ERK1/2, anti-phospho-Raf (Ser 259), anti-phospho-SHP-2 (Tyr 542), anti-phospho-STAT3 (Tyr 705), anti-phospho-JAK1 (Tyr 1022/1023), and anti-phospho-JAK3 (Tyr 980/981) (Cell Signaling), anti-SHP-2 (C18), anti-Raf-1 (E10), anti-JAK1 (HR785), and anti-STAT3 (C20) (Santa Cruz), and anti-JAK3 (Millipore), and anti-αtubulin (B5-1-2) (Sigma).
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