anti flag ha beads  (MedChemExpress)

Journal: Advanced Science

Article Title: Ferritinophagy Rewires Carnitine‐Dependent Lipid Metabolism to Inhibit PRRSV and IAV Replication

doi: 10.1002/advs.75721

Figure Lengend Snippet: PRRSV Nsp5 downregulates NCOA4 via ferritinophagy. (A) NCOA4 promoter activity was assessed by a dual‑luciferase reporter assay. MARC‐145 cells were co‐transfected with the reporter plasmid and a reference plasmid, followed by PRRSV infection. ns indicates not significant, Student's t ‐test, n ≥ 3. (B) MARC‐145 cells were infected with PRRSV (MOI = 1), and inhibitors BafA1 and CQ were added 12 h prior to sample collection. Immunoblotting analysis was used to detect NCOA4 protein expression. (Original blot images in Figure 7a). (C,D) Screening for PRRSV proteins that regulate NCOA4. (C) HEK‑293T cells were transfected with plasmids encoding empty vector, GP2, GP5, Nsp2, Nsp3, or Nsp5. (D) HEK‑293T cells were transfected with increasing amounts of an Nsp5‑Flag plasmid or the Flag‑empty vector control. Cells were harvested 36 h post‑transfection, and immunoblotting analysis was used to detect NCOA4 protein expression. (Original blot images in Figure 7b‐7c). (E) 3D structural representation illustrating the interaction among Nsp5 (yellow), NCOA4 (blue) and DDB1 (pink). (F) HEK‑293T cells were transfected to overexpress Nsp5, along with siRNA targeting DDB1 (siDDB1). Immunoblotting analysis was used to detect NCOA4 protein expression. (Original blot images in Figure 7d). (G–K) Co‑IP assays to examine interactions among Nsp5, DDB1, and NCOA4. (G,H) HEK‐293T cells were transfected with NCOA4‐MYC or Nsp5‐Flag along with DDB1‐HA or HA empty vector. (I) HEK‐293T cells were transfected with Flag empty vector and Nsp5‐Flag recombinant plasmids. (J,K) HEK‐293T cells were transfected with NCOA4‐MYC and Nsp5‐Flag or the corresponding empty vector. Cell lysates were collected 36 h post‐transfection, followed by affinity separation, and co‐immunoprecipitation analysis with specific antibodies. (Original blot images in Figure 7e‐7i). (L–O) HEK‐293T cells were co‐transfected with Flag empty vector or Nsp5‐Flag and NCOA4‐MYC along with HA‐Ub or HA‐K63, followed by affinity separation using anti‐MYC or anti‐Flag beads and co‐immunoprecipitation analysis with specific antibodies. (Original blot images in Figure 7j‐7m).

Article Snippet: The lysate was centrifuged at 12 000 × g for 10 min, and the supernatant was incubated at room temperature for 1 h with anti‐HA, anti‐Flag, or anti‐c‐Myc magnetic beads (HY‐K0201, HY‐K0207, HY‐K0206, MedChem Express, USA).

Techniques: Activity Assay, Reporter Assay, Transfection, Plasmid Preparation, Infection, Western Blot, Expressing, Control, Recombinant, Immunoprecipitation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: TRIM26-mediated regulation of TRAF6 ubiquitination enhances host immune response during Toxoplasma gondii infection

doi: 10.1007/s00018-026-06088-2

Figure Lengend Snippet: TRIM26 binds to TRAF6. ( A ) Verification of the overexpression efficiency of pcDNA3.1-TRAF6-HA plasmid by immunoblotting (IB). Ctrl: Untransfected HEK293T cells; Vector: HEK293T cells transfected with an empty vector (detection was performed using an anti-TRAF6 antibody). ( B ) Noninfected HEK293T cells were transfected with 3 µg of empty vector plasmid or pcDNA3.1-TRAF6-HA plasmid for 24 h. The TRAF6-containing complex was pulled down by immunoprecipitation (IP) using an anti-HA antibody. The immunoprecipitated products were subjected to SDS-PAGE, followed by liquid chromatography mass spectrometry (LC-MS) analysis. ( C ) Identification of protein names and unique peptides from the MS data. ( D ) HEK293T cells were transfected with the indicated plasmids. Cell lysates were subjected to IP with anti-Flag magnetic beads, followed by IB with anti-Flag and anti-Myc antibodies. ( E ) BMDMs were isolated from wild-type (WT) mice, cultured, and then used to prepare cell lysates. IP was performed using an anti-TRAF6 antibody or rabbit IgG antibody (as a negative control), and the immunoprecipitated products were analyzed by WB using anti-TRAF6 and anti-TRIM26 antibodies. ( F ) HEK293T cells transfected with TRIM26-Myc and TRAF6-Flag plasmids were fixed and stained with anti-Myc antibody (green) and an anti-Flag antibody (red). Additionally, BMDMs were stained with an anti-TRIM26 antibody (green) and an anti-TRAF6 antibody (red). The colocalization of TRIM26 and TRAF6 was visualized using confocal microscopy. Scale bars = 5 μm

Article Snippet: After centrifugation at 13,000 rpm for 30 min, the supernatants were collected and incubated overnight at 4 °C with anti-Flag magnetic Beads (HY-K0201A, MCE, China).

Techniques: Over Expression, Plasmid Preparation, Western Blot, Transfection, Immunoprecipitation, SDS Page, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Magnetic Beads, Isolation, Cell Culture, Negative Control, Staining, Confocal Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: TRIM26-mediated regulation of TRAF6 ubiquitination enhances host immune response during Toxoplasma gondii infection

doi: 10.1007/s00018-026-06088-2

Figure Lengend Snippet: TRIM26 attenuates the K48-linked ubiquitination of TRAF6. ( A ) HEK293T cells were co-transfected with TRIM26-Myc, TRAF6-Flag, and WT-Ubiquitin-HA plasmids for 36 h. Whole cell lysates (WCL) were prepared, followed by IP against TRAF6 using anti-Flag magnetic beads. The ubiquitination level of TRAF6 was detected by WB with an anti-HA antibody. ( B ) HEK293T cells were co-transfected with TRIM26-Myc, TRAF6-Flag, WT Ubiquitin-HA and various single-lysine ubiquitin mutants (K6, K11, K27, K29, K33, K48, and K63) for 36 h. Cell lysates were subjected to IP with anti-Flag magnetic beads, and TRAF6 ubiquitination was analyzed by WB using an anti-HA antibody. ( C ) HEK293T cells were co-transfected with TRIM26-Myc, TRAF6-Flag, WT-Ub-HA, K48-Ub-HA and K48R-Ub-HA plasmids for 36 h. Cell lysates were subjected to IP with anti-Flag beads, followed by WB analysis with an anti-HA antibody

Article Snippet: After centrifugation at 13,000 rpm for 30 min, the supernatants were collected and incubated overnight at 4 °C with anti-Flag magnetic Beads (HY-K0201A, MCE, China).

Techniques: Ubiquitin Proteomics, Transfection, Magnetic Beads