Immunoblotting was performed as previously described (20 (link)). For examination of the protein involved in pro-survival signaling pathway, cell lysis from empty-, JAK2-, or JAK2V617F-expressing macrophages were extracted and subpackaged equally for three groups (each group included empty, JAK2 and JAK2V617F lysates), followed by separation on 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. After semi-dry transfer, the membranes were sequentially probed with the indicated antibodies. Anti-p-JAK2 (T1007/1008) (#3776), anti-JAK2 (D2E12) (#3230), anti-p-STAT1 (T701) (#7649), anti-STAT1 (D1K9Y) (#14994), anti-p-STAT3 (Tyr705, D3A7) (#9145), anti-STAT3 (D3Z2G) (#12640), anti-p-STAT5 (Tyr694, C11C5) (#9359), anti-STAT5 (D206Y) -(#94205), anti-p-p38 (Thr180/Tyr182, D3F9) (#4511), anti-p38 (D13E1) (#8690), anti-p-AKT (Ser473, D9E) (#10019), anti-AKT (#9272), anti-p-JNK (Thr183/Tyr185, 81E11) (#4668), and anti-JNK (#9252) antibodies were purchased from Cell Signaling Technology. Anti-HK1 (19662-1-AP), anti-HK2 (22029-1-AP), anti-PKM1 (15821-1-AP), anti-flag (Sigma), and anti-β-actin (66009-1-Ig) antibodies were purchased from ProteinTech.
Anti stat1 d1k9y
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-STAT1 (D1K9Y) is a rabbit monoclonal antibody that binds to STAT1 protein. STAT1 is a member of the Signal Transducer and Activator of Transcription (STAT) protein family, which plays a key role in cellular signaling pathways. This antibody can be used for various applications, such as western blotting, immunoprecipitation, and immunohistochemistry, to detect and study the STAT1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti p stat1 y701, by Cell Signaling Technology (2 mentions)
Anti rabbit or anti mouse igg hrp conjugated antibody from sheep, by GE Healthcare (2 mentions)
Anti ifitm1, by Thermo Fisher Scientific (2 mentions)
Anti sars cov cov 2 spike 1a9, by GeneTex (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti jak2 d2e12, by Cell Signaling Technology (1 mentions)
Anti p38 d13e1, by Cell Signaling Technology (1 mentions)
Anti β actin 66009 1 ig, by Proteintech (1 mentions)
Anti jnk, by Cell Signaling Technology (1 mentions)
Ab28815, by Abcam (1 mentions)
Ab28829, by Abcam (1 mentions)
Ab32520, by Abcam (1 mentions)
Ecl substrate solution, by Merck Group (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti tbk1 nak d1b4, by Cell Signaling Technology (1 mentions)
Anti phospho tbk1 nak ser172 d52c2, by Cell Signaling Technology (1 mentions)
Anti irf3 d83b9, by Cell Signaling Technology (1 mentions)
Anti phospho stat1 tyr701 58d6, by Cell Signaling Technology (1 mentions)
6 protocols using anti stat1 d1k9y
1
Immunoblotting of JAK2 and STAT Signaling
2
STAT Expression and Phosphorylation in Lung Tissue
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The expression levels of STAT1, STAT4, STAT6 and the corresponding phosphorylated proteins in lung digests were analyzed by Western blot analysis. Cell lysates (40 μg) were separated by 10% sodium dodecyl sulfate-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Roche, USA). After incubation in a blocking buffer containing 5% skim milk in TBST (12.5 mM Tris–HCl pH 7.5, 68.5 mM NaCl, and 0.1% Tween 20) for 1 h, the blots were incubated overnight with primary antibodies including rabbit anti-STAT1 (D1K9Y, Cell Signaling Technology, USA), rabbit anti-phosphorylated STAT1 (Tyr701, Cell Signaling Technology, USA), rabbit anti-STAT4 (C46B10, Cell Signaling Technology, USA), rabbit anti-phosphorylated STAT4 (ab28815, Abcam, UK), rabbit anti-STAT6 (ab32520, Abcam, UK), and rabbit anti-phosphorylated STAT6 (ab28829, Abcam, UK). An anti-mouse GAPDH antibody was used as a loading control. The blots were washed with TBST, incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit Ig (Jackson ImmunoResearch) and then developed with an enhanced chemiluminescence (ECL) substrate solution (Millipore).
3
Immunoblotting Analysis of Innate Immune Signaling
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Primary antibodies used: Anti-IRF3 (D83B9) Cat# 4302 (Cell Signaling); anti-phospho- IRF3 (Ser396) (D601M) Cat#29047 (Cell Signaling); anti-STAT1 (D1K9Y) Cat# 14994 (Cell Signaling); anti-phospho-STAT1 (Tyr701) (58D6) Cat#9167 (Cell Signaling): anti- STING (D2P2F) Cat# 13647 (Cell Singaling); anti-TBK1/NAK (D1B4) Cat#3504 (Cell Signaling); anti-phospho-TBK1/NAK (Ser172) (D52C2) Cat#5483 (Cell Signaling); Anti-Actin (Sigma).
4
Protein Extraction and Immunoblot Analysis of Organoids
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Organoids were collected using a cell recovery solution (Corning) and washed with PBS, and proteins were extracted using 1% NP‐40 buffer (20 mmol/L Tris‐HCl, pH 8.0, 137 mmol/L NaCl, 1% NP‐40, 10% glycerol). The protein concentrations were determined using a protein assay (Bio‐Rad, Hercules, CA, USA), and equal amounts of protein were resolved, transferred to nitrocellulose membranes and detected as previously described.11 The antibodies used for the immunoblot analysis were as follows: anti‐STAT1(D1K9Y, Cell Signaling), anti‐phosphoSTAT1 (Ser727) (D3B7, Cell Signaling, Danvers, MA, USA), anti‐phosphoSTAT1 (Tyr701) (58D6, Cell Signaling), anti‐ERK (137F5, Cell Signaling), anti‐phosphoERK (Thr202/Tyr204) (D13.14.4E, Cell Signaling), anti‐S6 (5G10, Cell Signaling), anti‐phosphoS6 (Ser235/236) (D57.2.2E, Cell Signaling), and anti‐actin (AC‐15, Sigma‐Aldrich).
5
Western Blot Analysis of Viral and Host Proteins
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Viral and host protein analysis were evaluated as previously described (55 (link), 59 (link)). Briefly, cell lysates were resolved on 7.5% Mini-Protean TGX SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes using a Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked with 5% (wt/vol) nonfat dry milk in TBST (TBS plus 0.1% [vol/vol] Tween 20) for 1 h and then probed with the indicated primary antibody in 3% (wt/vol) bovine serum albumin in TBST at 4°C overnight. After overnight incubation, the membranes were probed with the following secondary antibodies in 5% (wt/vol) nonfat dry milk in TBST for 1 h at room temperature: anti-rabbit or anti-mouse IgG-HRP-conjugated antibody from sheep (both 1:10,000; GE Healthcare). Proteins were visualized using ECL or SuperSignal West Femto chemiluminescence reagents (Pierce) and detected by autoradiography. The following primary antibodies were used: anti-pSTAT1 Y701 (1:1,000; Cell Signaling Technologies, 9171L), anti-STAT1 D1K9Y (1:1,000; Cell Signaling Technologies, 14994P), anti-IFITM1 (1:1,000; Invitrogen, PA5-20989), anti-SARS-CoV/CoV-2 Spike 1A9 (1:1,000; GeneTex, GTX632604), and anti-β-actin (1:1,000; Abcam, ab8227).
6
SARS-CoV-2 Spike Protein Western Blot Analysis
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Viral and host protein analysis were evaluated as previously described (52 , 56 ). Briefly, cell lysates were resolved on 7.5% Mini-PROTEAN TGX SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes using a Trans-Blot Turbo transfer system (BioRad). Membranes were blocked with 5% (w/v) non-fat dry milk in TBST (TBS with 0.1% (v/v) Tween-20) for 1 hr, and then probed with the indicated primary antibody in 3% (w/v) BSA in TBST at 4°C overnight. Following overnight incubation, membranes were probed with the following secondary antibodies in 5% (w/v) non-fat dry milk in TBST for 1 hr at room temperature: anti-rabbit or anti-mouse IgG-HRP conjugated antibody from sheep (both 1:10,000 GE Healthcare). Proteins were visualized using ECL or SuperSignal West Femto chemiluminescence reagents (Pierce) and detected by autoradiography. The following primary antibodies were used: anti-pSTAT1 (Y701) (1:1000 9171L Cell Signaling Technologies), anti-STAT1 D1K9Y (1:1000 14994P Cell Signaling Technologies), anti-IFITM1 (1:1000 PA5–20989 Invitrogen), anti-SARS-CoV/CoV-2 Spike 1A9 (1:1000 GTX632604 GeneTex), and anti-β-Actin (1:1000 ab8227 Abcam).
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