Glomerular or cortical samples were sonicated and/or mechanically disrupted and used for sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) as previously reported 33 (link). We used the following primary antibodies from Cell Signaling Technology: Jak2 (D2E12), Stat3, phospho-Stat3 (Tyr705) and β-actin. The fibronectin antibody was from EMD Millipore. The secondary antibodies were from Santa Cruz Biotechnology. All exposures were within the linear range of the film and normalized to β-actin content whenever feasible.
Jak2 d2e12
Manufactured by Cell Signaling Technology
Sourced in United States
Jak2 (D2E12) is a rabbit monoclonal antibody that recognizes the Janus Kinase 2 (JAK2) protein. JAK2 is a member of the Janus kinase family of non-receptor tyrosine kinases and plays a key role in cytokine and growth factor signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Jak1 6g4, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
P jak2, by Cell Signaling Technology (1 mentions)
Tyr1008 d4a8, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Phospho p38 map kinase, by Cell Signaling Technology (1 mentions)
Ab109235, by Abcam (1 mentions)
P38mapk d13e1, by Cell Signaling Technology (1 mentions)
Pi3 kinase p85 19h8, by Cell Signaling Technology (1 mentions)
β actin c4, by Santa Cruz Biotechnology (1 mentions)
Fusion fx system, by Avantor (1 mentions)
Stat1, by Cell Signaling Technology (1 mentions)
Ecl prime western blotting detection kit, by GE Healthcare (1 mentions)
Whatman, by Cytiva (1 mentions)
7 protocols using jak2 d2e12
1
Immunoblotting of Kidney Samples
2
SDS-PAGE and Western Blot Analysis
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Cell lysis, SDS-PAGE and Western blots were performed as described previously.31 (link) For pJAK2 analysis cells were lysed directly in 1 × Laemmli sample buffer. Antibodies used were: pSTAT3 (Tyr705, CS#9131), STAT3 (CS#9132S), STAT4 (clone C46B10, CS#2653), pJAK2 (Tyr1008, D4A8, CS#8082), JAK2 (D2E12, CS#3230), GzmB (CS#4275) and Prf1 (CS#3693) all from Cell Signaling Technology; panERK (clone 16/ERK) and pSTAT4 (Tyr693, clone 38/pSTAT4) from BD Transduction Laboratories. Quantification of Western blots was done with ImageJ software.
3
OT-I Adoptive Transfer Protocol
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OT-I mice and OT-I mice deficient for IL-12 receptor β1 (gifts from Dr.
Mescher, University of Minnesota) have a transgenic TCR specific for H-2Kb and OVA257–2649 (link),10 (link),73 (link). Mice were maintained under specific
pathogen-free conditions at the University of Maryland, and these studies have been
reviewed and approved by the Institutional Animal Care and Use Committee. C57BL/6 male
mice were purchased from the National Cancer Institute. Vaccinia virus preferentially
accumulates in ovaries 74 (link), 75 (link), which may cause variation in CTL activation
under different treatments. As a result, only male recipient mice were used for vaccinia
virus infection. Phospho-Stat4 (Tyr693) (D2E4) and Jak2 (D2E12) were purchased from Cell
signaling Technology (Danvers, MA). All the rest directly conjugated fluorescent
antibodies were purchased from BD Biosciences, eBioscience or Biolegend. Rapamycin were
purchased from EMD (Gibbstown, NJ). Kb/OVA tetramer is a gift from Dr. Jameson
from University of Minnesota.
Mescher, University of Minnesota) have a transgenic TCR specific for H-2Kb and OVA257–2649 (link),10 (link),73 (link). Mice were maintained under specific
pathogen-free conditions at the University of Maryland, and these studies have been
reviewed and approved by the Institutional Animal Care and Use Committee. C57BL/6 male
mice were purchased from the National Cancer Institute. Vaccinia virus preferentially
accumulates in ovaries 74 (link), 75 (link), which may cause variation in CTL activation
under different treatments. As a result, only male recipient mice were used for vaccinia
virus infection. Phospho-Stat4 (Tyr693) (D2E4) and Jak2 (D2E12) were purchased from Cell
signaling Technology (Danvers, MA). All the rest directly conjugated fluorescent
antibodies were purchased from BD Biosciences, eBioscience or Biolegend. Rapamycin were
purchased from EMD (Gibbstown, NJ). Kb/OVA tetramer is a gift from Dr. Jameson
from University of Minnesota.
4
Analyzing Protein Signaling Cascades
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The CMECs were homogenized in RIPA lysis buffer (#P0013C; Beyotime) containing 1X Phosphatase Inhibitor Cocktail (#5870S; Cell Signaling Technology, Beverly, MA, USA) and 1 µg/ml each of aprotinin (A1153; Sigma-Aldrich) and leupeptin (#L2884; Sigma-Aldrich). Forty micrograms of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and then probed with antibodies for XO (#ab109235; Abcam), phospho-PI3 kinase p85 (Tyr458)/p55 (Tyr199) (#4228), PI3 kinase p85 (19H8) (#4257), phospho-p38 MAP kinase (Thr180/Thr182) (#4631), p38 MAPK (D13E1) (#8690), JAK2 (D2E12) (#3230) and phospho-JAK2 (Tyr1007/1008) (C80C3) (#3776) (all from Cell Signaling Technology, Beverly, MA, USA). The same membranes were reprobed with an antibody for tubulin (#AT819; Beyotime). The blotting film was quantified using a scanner and a densitometry program (ImageJ; https://imagej.nih.gov/ij/index.html ) (24 ). To quantify the phosphor-specific signal in the activated samples, the background was subtracted and the band was normalized to the amount of tubulin or total target protein in the lysate.
5
Western Blotting of Immune Regulators
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Cells were lysed in 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100, and protease inhibitor (cOmplete; Roche). 20 µg of total protein extracts were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Optitran BA-S 85 Reinforced NC; Whatman). The membrane was incubated in PBS supplemented with 0.4% Tween-20 and 5% dry milk for 1 h. Primary antibodies recognizing Jak1 (6G4; Cell Signaling Technology), Jak2 (D2E12; Cell Signaling Technology), Stat1 (polyclonal; Cell Signaling Technology), Phospho-Stat1 (58D6 [Tyr701]; Cell Signaling Technology), ERAP1 (6H9), MECL (K6512), LMP2 (polyclonal; Abcam), LMP7 (polyclonal; Abcam), or β-Actin (C4; Santa Cruz Biotechnology, Inc.) were diluted in PBS/0.1% Tween-20 and 2.5% dry milk and were applied overnight at 4°C. The membrane was washed three times with PBS/0.1% Tween-20 followed by 1-h incubation with the secondary Ab (0.2 µg/ml goat anti–rabbit IgG H&L chain–specific peroxidase conjugate, rabbit anti–mouse IgG H&L chain–specific peroxidase conjugate, or rabbit anti–goat IgG H&L chain–specific peroxidase conjugate; EMD Millipore). The ECL Prime Western Blotting Detection kit (GE Healthcare) was used for detection. Membranes were analyzed with the Fusion FX System using FusionCapt Advance FX7 Software (Peqlab).
6
Western Blot Analysis of Signaling Proteins
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Sample preparation of whole cell lysates, SDS-PAGE, membrane transfer and blotting were performed according to standard protocols. Antibodies to p21 (#2947), PTPN2 (TCPTP) (#58935), Jak2 (D2E12) (#3230), Phospho-Jak2 (Tyr1007/1008) (#3771), Stat3 (D1B2J) (#30835), phospho-Stat3 (Tyr705) (D3A7) (#9145), Stat5 (D3N2B) (#25656), phospho-Stat5 (Tyr694) (D47E7) (#4322), MDM2 (D1V2Z) (#86934), α-Tubulin (#2144) and GAPDH (#5174) were purchased from Cell Signaling Technologies. Antibody to TP53 (sc-47698) and MDMX (G-10) (sc-74467) were purchased from Santa Cruz Biotechnology. Antibodies were used at 1:1000 dilution (Cell Signaling Technologies) or at 1:500 dilution (Santa Cruz Biotechnology).
Uncropped western blots from the main figures are provided in Supplementary Figure8 .
Uncropped western blots from the main figures are provided in Supplementary Figure
7
Alisertib Modulates Aurora A and Jak/STAT Signaling
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The effect of alisertib on Aurora A and the Jak/STATs signaling pathways was assessed by immunoblotting, as described previously 21 (link), 23 , using the following phospho-specific antibodies: p-Aurora A/B/C (Cell Signaling Technology, #2914), Aurora A (35C1) (Abcam, ab13824), p-Jak1 (T1022/1023) (Cell Signaling Technology, #3331), Jak1 (6G4) (Cell Signaling Technology, #3344), p-Jak2 (T1007/1008) (Cell Signaling Technology, #3776), Jak2 (D2E12) (Cell Signaling Technology, #3230), p-STAT1 (T701) (Cell Signaling Technology, #7649), STAT1 (D1K9Y) (Cell Signaling Technology, #14994), p-STAT2 (T690) (Cell Signaling Technology, #4441), STAT2 (D9J7L) (Cell Signaling Technology, #72604), PARP (46D11) (Cell Signaling Technology, #9532), DNMT3B (D7070) (Cell Signaling Technology, #67259), DNMT1 (Proteintech., 24206-1-AP), DNMT3A (Proteintech., 19366-1-AP), ubiquitin (Proteintech., 10201-1-AP), UHRF1(Proteintech., 21402-1-AP), flag (Abmart, M20008L) and β-actin (Proteintech.,66009-1-Ig).
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