beclin 1  (MedChemExpress)

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: The interaction between NS1 SC09 and LRPPRC results in initiation of BECN1-dependent autophagy. (A) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP. (B) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP. (C) 293T cells were transfected with the indicated plasmids above. Cell lysates were subjected to Flag-IP. (D) 293T cells were transfected with empty vector, NS1 SC09 -HA, NS1 JL89 -HA, or NS1 WSN -HA. Cell lysates were prepared and immunoprecipitated with the anti-BECN1 antibody or control IgG. (E) The indicated plasmids above were co-transfected into 293T cells. Cell lysates were subjected to Flag-IP. (F) 293T cells were transfected with empty vector, NS1 SC09 -HA, NS1 JL89 -HA, or NS1 WSN -HA. Cell lysates were prepared and immunoprecipitated with the anti-PIK3C3 antibody or control IgG. (G) The indicated plasmids above were co-transfected into 293T cells. Cell lysates were subjected to Flag-IP.

Article Snippet: For immunoprecipitation and western blotting, infected or transfected cells were lysed using ice-cold lysis buffer (50 mM Tris-HCl [Biosharp, 0234]), pH 7.5, 50 mM NaCl [Sigma-Aldrich, S7653], 5 mM EDTA [Sigma-Aldrich, E6758]), 1% Triton X-100 [Sigma-Aldrich, T7878]) and centrifuged at 13,000 × g and 4°C for 10 min. After centrifugation, the crude lysates were incubated with anti-FLAG M2 magnetic beads (Sigma-Aldrich, M8823), anti-HA magnetic beads (MCE, HY-K0201), or protein A/G magnetic beads (MCE, HY-K0202) bound with anti-BECN1 antibody or anti-PIK3C3 antibody at 4°C for 2 h. After incubation, the beads were collected using a magnetic separator and washed three times with PBS.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Control

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: BECN1 plays a key role in the autophagy pathway induced by NS1 SC09 . (A) 293T cells were stimulated with starvation or treated with Eug or Cal, and BECN1 KO cells were over-expressed with BECN F123A -Flag as indicated. The cells mentioned above were transfected with indicated plasmids above. Cell lysates were subjected to Flag-IP. (B and C) 293T cells, BECN1 KO cells, and BECN1 KO cells overexpressing of BECN1-Flag or Becin1 F123A -Flag as indicated above were infected with WSN, WSN-NS1 SC09 , or WSN-NS1 JL89 at the MOI of 5.0 and 0.01. The supernatants were sampled at 24 h post-infection, and the viral titers were determined with endpoint titration in MDCK cells. The cell lysates were prepared and analyzed using western bloting. (D-G) 293T cells and A549 cells were infected with WSN, WSN-NS1 SC09 , or WSN-NS1 JL89 after stimulation with starvation or treatment with Eug or Cal as indicated above. The supernatants were sampled at 24 h post-infection, and the viral titers were determined using endpoint titration in MDCK cells. The cell lysates were prepared and analyzed using western blotting. Error bars indicate the SD from three independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: For immunoprecipitation and western blotting, infected or transfected cells were lysed using ice-cold lysis buffer (50 mM Tris-HCl [Biosharp, 0234]), pH 7.5, 50 mM NaCl [Sigma-Aldrich, S7653], 5 mM EDTA [Sigma-Aldrich, E6758]), 1% Triton X-100 [Sigma-Aldrich, T7878]) and centrifuged at 13,000 × g and 4°C for 10 min. After centrifugation, the crude lysates were incubated with anti-FLAG M2 magnetic beads (Sigma-Aldrich, M8823), anti-HA magnetic beads (MCE, HY-K0201), or protein A/G magnetic beads (MCE, HY-K0202) bound with anti-BECN1 antibody or anti-PIK3C3 antibody at 4°C for 2 h. After incubation, the beads were collected using a magnetic separator and washed three times with PBS.

Techniques: Transfection, Infection, Titration, Western Blot

Journal: Autophagy

Article Title: A/(H1N1) pdm09 NS1 promotes viral replication by enhancing autophagy through hijacking the IAV negative regulatory factor LRPPRC

doi: 10.1080/15548627.2022.2139922

Figure Lengend Snippet: A proposed model for the pathway of autophagy induced by NS1 pdm09 . In the normal physiological state, LRPPRC interacts with the BECN1-BCL2 heterodimer, forming a stable complex in the mitochondria, thereby inhibiting BECN1-dependent autophagy. During A/(H1N1) pdm09 infection, the NS1 pdm09 promotes viral replication by enhancing autophagy which is mainly attributed to the inhibitory effect of NS1 pdm09 on the negative regulation of autophagy by LRPPRC. The interaction between NS1 pdm09 and LRPPRC competitively blocks the interaction of LRPPRC with BECN1-BCL2 heterodimer, resulting in degradation of BCL2 and increased recruitment of BECN1 by PIK3C3, and then induction of the initiation of autophagy.

Article Snippet: For immunoprecipitation and western blotting, infected or transfected cells were lysed using ice-cold lysis buffer (50 mM Tris-HCl [Biosharp, 0234]), pH 7.5, 50 mM NaCl [Sigma-Aldrich, S7653], 5 mM EDTA [Sigma-Aldrich, E6758]), 1% Triton X-100 [Sigma-Aldrich, T7878]) and centrifuged at 13,000 × g and 4°C for 10 min. After centrifugation, the crude lysates were incubated with anti-FLAG M2 magnetic beads (Sigma-Aldrich, M8823), anti-HA magnetic beads (MCE, HY-K0201), or protein A/G magnetic beads (MCE, HY-K0202) bound with anti-BECN1 antibody or anti-PIK3C3 antibody at 4°C for 2 h. After incubation, the beads were collected using a magnetic separator and washed three times with PBS.

Techniques: Infection