Cells were washed with phosphate buffered saline (PBS) and incubated in 100 μl of RIPA cell lysis buffer (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) for 20 minutes. They were then snap-frozen in liquid nitrogen, thawed, and centrifuged at 125g for 10 minutes. The supernatant was collected, and assayed for protein concentration using the spectrophotometric Bradford protein Assay (Bio-Rad). Approximately 30 μg of proteins were electrophoresed in 10% SDS-PAGE gel, and transferred on a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% non-fat blocking grade milk (Bio-Rad) and probed with a 1:1000 dilution of primary antibody overnight. The following antibodies were used: G-CSF receptor (Abcam), JAK-2 (Abcam), phospho-JAK2 (Abcam), PI3K (Cell Signaling Technology, Danvers, MA), phospho-PI3K (Cell Signaling Technology), Akt (Cell Signaling Technology), phospho-Akt (Cell Signaling Technology), PDE3B (Abcam), and actin (Santa Cruz Biotechnology). After washing three times, the membranes were probed with a 1:1000 dilution of secondary antibodies (Santa Cruz Biotechnology) for 1 hour at room temperature. The protein bands were visualized with ECL Plus, Chemiluminescence (GE Healthcare and Life Sciences, Piscataway, NJ). The densities were analyzed using Image J Software (Version 1.43u; National Institutes of Health, Bethesda, MD) and normalized to actin.
Phospho jak2
Manufactured by Abcam
Sourced in United States
Phospho-JAK2 is a lab equipment product that detects the phosphorylation of the JAK2 protein. JAK2 is a tyrosine kinase that plays a crucial role in various cellular signaling pathways. The Phospho-JAK2 product can be used to analyze the activation state of JAK2 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Phospho stat3, by Abcam (3 mentions)
Ab 2128522, by Cell Signaling Technology (2 mentions)
Ab 331269, by Cell Signaling Technology (2 mentions)
Ab 775808, by Abcam (2 mentions)
Ab 1658549, by Abcam (2 mentions)
Odyssey two color infrared scanner, by LI COR (2 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
β actin, by Bioworld Technology (2 mentions)
Actin, by Santa Cruz Biotechnology (1 mentions)
Non fat blocking grade milk, by Bio-Rad (1 mentions)
Phospho pi3k, by Cell Signaling Technology (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Ripa cell lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Ab 2070042, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Actin, by Merck Group (1 mentions)
P70s6k, by Santa Cruz Biotechnology (1 mentions)
4e bp1, by Thermo Fisher Scientific (1 mentions)
7 protocols using phospho jak2
1
Western Blot Analysis of Cell Signaling Pathways
2
Western Blot Analysis of Myocardial Proteins
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The JAK2, STAT3, pJAK2, pSTAT3, and cleaved caspase-3 proteins of myocardial tissue were extracted as per kit indications. The protein concentration was quantified. Western blot analysis was performed as previously described (11 (link)). Primary antibodies against the following antigens were used: JAK2 (1:1,000, AB_2128522; Cell Signaling Technology, Danvers, MA, USA), STAT3 (1:1,000, AB_331269; Cell Signaling Technology), phospho-JAK2 (phosphor-Y1007 + Y1008, 1:1,000, AB_775808; Abcam, Cambridge, UK), phospho-STAT3 (Tyr705, 1:1,000, AB_1658549; Abcam), β-actin (1:3,000, Bioworld Technology, Minneapolis, MN, USA). The secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG (1:10,000, Cell Signaling Technology). The membranes were detected with an Odyssey two-color infrared scanner (LI-Cor Biosciences, Lincoln, NE, USA).
3
Western Blot Analysis of JAK-STAT Signaling
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For Western blot analysis, proteins from different groups were extracted and protein concentration quantified. Then, samples were used for routine Western blot analysis; the detailed protocol has been described previously.17 (link) Antibodies were replaced by the primary antibodies JAK2 (1:1,000, AB_2128522; Cell Signaling Technology, Danvers, MA, USA), phospho-JAK2 (phospho-Y1007 + Y1008, 1:1,000, AB_775808; Abcam, Cambridge, UK), STAT3 (1:1,000, AB_331269; Cell Signaling Technology), phospho-STAT3 (Tyr705, 1:1,000, AB_1658549; Abcam), cleaved caspase 3 (1:1,000, AB_2070042; Cell Signaling Technology), and β-actin (1:3,000; Bioworld Technology, Minneapolis, MN, USA). The secondary antibody was horseradish peroxidase conjugated to goat antirabbit/mouse IgG (1:10,000; Cell Signaling Technology). The membranes were developed using an Odyssey two-color infrared scanner (LI-Cor Biosciences, Lincoln, NE, USA).
4
Western Blot Analysis of Polycystic Kidney Proteins
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The following primary antibodies were used for Western blot analysis: Polycystin‐2 (sc‐10376 Santa Cruz Biotechnology), p70‐S6K (sc‐230 Santa Cruz Biotechnology), phospho‐p70‐S6K (sc‐8416 Santa Cruz Biotechnology), phospho‐mTOR (5536 CST), phospho‐4E‐BP1 (2855 CST), PTEN (9559 CST), Akt (9272 CST), phospho‐Akt (9271 CST), actin (MAB1501 Millipore), polycystin‐1 CT2741 (kindly provided by the Baltimore Polycystic Kidney Disease Research and Clinical Core Center), mTOR (701483 Thermo Fisher Scientific), 4EBP1 (AHO1382 Thermo Fisher Scientific), JAK2 (ab37226 Abcam), phospho‐JAK2 (ab32101 Abcam). The following secondary antibodies were used: goat anti‐mouse IgG HRP‐conjugate (AP124P Millipore), goat anti‐rabbit IgG HRP‐conjugate (AP132P Millipore), donkey anti‐goat IgG HRP‐conjugate (A00178 GenScript). The IgPKD1 inhibitory antibody was a generous gift from Dr O. Ibraghimov‐Beskrovnaya and H. Husson (Genzyme Co., Boston).
5
Antibodies for JAK/STAT Signaling Analysis
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The antibodies that were used in this study were PhosphoJAK2 (Tyr1007, Tyr1008), JAK1 antibody (EPR349[N]) (ab133666), and Phospho-JAK1 (phospho-Y1022 + Y1023) antibody (EPR1899[2 (link)]) (ab138005) (Abcam, Cambridge, UK); Phospho-Jak2 [p Tyr1007, p Tyr1008] antibody (SY24-03), Phospho-STAT1 [p Tyr701] antibody, STAT3 (232209) antibody, and Phospho-STAT3 (Tyr705) antibody (1004G) (Novus Bio., Cambridge, UK); SOCS7 antibody (PA5-44102), JAK2 monoclonal antibody (691R5), and actin monoclonal antibody (ACTN05 [C4]) (Thermo Fisher Scientific). Secondary horseradish peroxidase (HRP)-linked anti-mouse and anti-rabbit IgG antibodies were supplied in the Western Breeze™ Chemiluminescent Kit (Thermo Fisher Scientific).
6
Quantification of Jak-Stat Signaling Proteins
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Proteins for western blot analysis were extracted from the mouse joint synovial tissue and lysed using RIPA Lysis Buffer which contained protease inhibitor cocktail (Applygen Technologies Inc., BeiJing). The protein concentration was quantified preliminarily with the BCA kit. The total proteins were separated using 7.5% SDS-PAGE and then imprinted on NC membrane. They were blocked for 1 h at room temperature using TBST containing 5% skimmed milk. The primary antibodies including anti-mouse Stat-6, Jak-2, Jak-3, phospho-Jak-2, phospho-Jak-3, and phospho-Stat-6 polyclonal antibodies (Abcam, Cambridge, MA, United States) were incubated overnight at 4°C. Next, the membranes were incubated by secondary antibodies (1:2000, Abcam, Cambridge, MA, United States) for 1 h and were treated with ECL chemiluminescence reagents. Three replicates of each experiment were performed. Densitometry plots showing protein expression were analyzed by ImageJ (Bethesda, United States). The housekeeping gene GAPDH was used as an internal reference.
7
Western Blot Analysis of Apoptosis Markers
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Cells were lysed using the CHAPS buffer, and the protein concentrations were measured using the Bradford assay kit (Beyotime). An equal amount of protein (20 μg) was resolved on 10% SDS‐PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Next, the PVDF membranes were blocked with skimmed milk for 1 h at room temperature, following which the membranes were incubated with primary antibodies overnight at 4°C. Afterward, the membranes were incubated with secondary horseradish peroxidase (HRP)‐conjugated antibodies (Sigma‐Aldrich). Immunoprecipitation was performed as described previously to detect the activation of Bax.8 The following primary antibodies were used: Caspase‐3 (CST, USA), Bcl‐2 (CST), Smac/DIABLO (CST), Cytochrome c (CST), Bax (CST), Bax (6A7) (CST), GRP78 (Abcam), CHOP (Abcam), phospho‐JAK2 (Abcam), phospho‐STAT3 (Abcam), and GAPDH (Sigma‐Aldrich). Secondary antibodies were obtained from Sigma‐Aldrich.
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