Western blotting was performed as previously described69 (link). Briefly, lysate samples were separated on 5–20% gradient polyacrylamide gel by SDS–PAGE following electrical transfer to PVDF membranes (GE Healthcare). After blocking with 5% dry milk, membranes were probed with the appropriate primary antibodies diluted in Immunoshot Reagent 1 (Cosmo bio, Tokyo, Japan) overnight at 4 °C. HRP-conjugated corresponding secondary antibodies (GE Healthcare) were subsequently used. Trueblot anti-rabbit IgG HRP (Rockland, Limerick, PA, USA, 1:1,000) was used as the secondary antibody to avoid interfering immunoprecipitated immunoglobulin heavy and light chains. Bound antibodies were detected using Immunostar LD reagents (Wako). The following antibodies were used: Hnrnp-U (#ab180952, 1:1,000) and Nucleolin (#ab22758, 1:1,000) from Abcam (Cambridge, UK); HA tag (#561, 1:10,000) and Syncrip (#RN046PW, 1:1,000) from MBL; Hnrnp-A2/B1 (#R4653, 1:1,000) and β-actin (#A1978, 1:2,000) from Sigma-Aldrich; and YBX1 (D299, 1:1,000), Igf2BP1 (D33A2, 1:1,000) and HnrnpA1 (D21H11, 1:1,000) from Cell Signaling Technology (CST, Danvers, MA, USA).
Hnrnp a1 d21h11
Manufactured by Cell Signaling Technology
Sourced in United States
HnRNP A1 (D21H11) is a rabbit monoclonal antibody that recognizes the HnRNP A1 protein. HnRNP A1 is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family and is involved in the regulation of gene expression and mRNA processing.
Automatically generated - may contain errors
Lab products found in correlation
Hnrnp u, by Abcam (1 mentions)
Trueblot anti rabbit igg hrp, by Rockland Immunochemicals (1 mentions)
Immunoshot reagent 1, by Cosmo Bio (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
β actin, by Merck Group (1 mentions)
Nucleolin, by Abcam (1 mentions)
Mettl3 e3f2a rabbit mab, by Cell Signaling Technology (1 mentions)
Foxo1 c29h4, by Cell Signaling Technology (1 mentions)
Protein g beads, by Thermo Fisher Scientific (1 mentions)
Protein g beads, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Negative isolation kit, by Miltenyi Biotec (1 mentions)
4 protocols using hnrnp a1 d21h11
1
Western Blotting Procedure for Protein Analysis
2
Detection of COVID-19 Biomarkers using Western Blotting
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N6-Methyladenosine (m6A) (D9D9W) Rabbit mAb, METTL3 (E3F2A) Rabbit mAb and hnRNP A1 (D21H11) Rabbit mAbs were received from Cell Signalling Technology (Massachusetts, USA). eIF4E monoclonal antibody (5D11) and Phospho-eIF4E (Ser209) polyclonal antibodies were received from Invitrogen (South San Francisco, CA, USA). Mouse anti-GAPDH (Glyceraldehyde 3-phosphate dehydrogenase, house-keeping control protein) primary antibody, Anti-Mouse IgG (whole molecule)−Alkaline Phosphatase antibody (produced in goat) and Anti-Rabbit IgG (whole molecule)–Peroxidase antibody (produced in goat) was received from Sigma-Aldrich (St. Louis, USA). Rabbit anti-human IgG–HRP was procured from GeNei™, Peenya (Bangalore, India). Human serum from a COVID-19 confirmed patient was received from the civil hospital, Hisar (Haryana).
3
Western Blot Antibody Analysis
4
CD4+ T Cell Immunoprecipitation Assay
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CD4+ cells were isolated using a negative isolation kit (Miltenyi Biotec) from WT and A1-MUT mice. 2–2.5 × 105 CD4+ T cells were plated in a 96-well plate (Falcon REF 353077) with (1.0 µg/ml) plate-bound anti-CD3 in the presence of soluble (1.0 µg/ml) anti-CD28 for 30 min. The cells were lysed in ice-cold lysis buffer (Cell Signaling) with protease inhibitor (Thermo Fisher) on ice for 40 min. The supernatant from the cell lysate was precleared with protein G beads (Thermo Fisher) for 30 min. Lysates were incubated with anti-hnRNP A1 (D21H11; Cell Signaling) Ab at 4°C overnight. The hnRNP A1 complexes were captured by protein G–coated beads. As a negative control, cell lysate treated with IgG (G3A1; Cell Signaling) Ab was incubated with protein G beads. The beads were washed four times with cell lysis buffer. The captured protein was eluted with SDS and heated at 95°C for 5 min. Western blotting was performed using an Ab that recognizes P-Akt RXRXXS/T (23C8D2; Cell Signaling), and imaged. Then, the same blot was stripped and probed for hnRNP A1(D21H11; Cell Signaling) for quantification.
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