Purification:Article Title: Molecular functions of the double-sided and inverted ubiquitin-interacting motif found in Xenopus tropicalis cryptochrome 6.
Article Snippet: .. The purified GST-fusion protein (3 μg) was mixed with polyubiquitin (2.2 μg, K48Ub2–8, UBPBio, D1500) in binding buffer (25 mM HEPES[pH 7.2], 125 mM CH3COOK, 2.5 mM CH3COOMg, 5 mM EDTA, 0.5% TritonX-100, 1 mM dithiothreitol, 1 cOmplete protease inhibitor cocktail (Rosche, 4693116001) and incubated with rotation for 30 min at 4 C. Then, the sample was mixed with glutathione-conjugated magnetic beads (MagneGST Glutathione Particles, Promega, V8611) and incubated with rotation for 1 h at 4 C. After unbound materials were washed out with the binding buffer, the precipitants were eluted with 50 mM glutathione (reduced form) in the binding buffer (pH 7.69) and analyzed by immunoblot analysis (Watari et al., 2012) using anti-ubiquitin polyclonal antibody (Proteintech, 102021-2-AP) and anti-GST monoclonal antibody (B-14, Santa Cruz Biotechnology, sc-138). .. In competition experiments, 2.2 μg of the polyubiquitin was mixed with 1 μg of synthetic polypeptide (XtCRY6DI-UIM_WT, ADLETLGYETDLELAIALSLQEHNQLTD or XtCRY6DI-UIM_SFL, ADLETLLEYGADLITAQESELLHNQLTD, Genscript) and incubated for 1 h at 4 C prior to the incubation with the GST-fusion protein.
Binding Assay:Article Title: Molecular functions of the double-sided and inverted ubiquitin-interacting motif found in Xenopus tropicalis cryptochrome 6.
Article Snippet: .. The purified GST-fusion protein (3 μg) was mixed with polyubiquitin (2.2 μg, K48Ub2–8, UBPBio, D1500) in binding buffer (25 mM HEPES[pH 7.2], 125 mM CH3COOK, 2.5 mM CH3COOMg, 5 mM EDTA, 0.5% TritonX-100, 1 mM dithiothreitol, 1 cOmplete protease inhibitor cocktail (Rosche, 4693116001) and incubated with rotation for 30 min at 4 C. Then, the sample was mixed with glutathione-conjugated magnetic beads (MagneGST Glutathione Particles, Promega, V8611) and incubated with rotation for 1 h at 4 C. After unbound materials were washed out with the binding buffer, the precipitants were eluted with 50 mM glutathione (reduced form) in the binding buffer (pH 7.69) and analyzed by immunoblot analysis (Watari et al., 2012) using anti-ubiquitin polyclonal antibody (Proteintech, 102021-2-AP) and anti-GST monoclonal antibody (B-14, Santa Cruz Biotechnology, sc-138). .. In competition experiments, 2.2 μg of the polyubiquitin was mixed with 1 μg of synthetic polypeptide (XtCRY6DI-UIM_WT, ADLETLGYETDLELAIALSLQEHNQLTD or XtCRY6DI-UIM_SFL, ADLETLLEYGADLITAQESELLHNQLTD, Genscript) and incubated for 1 h at 4 C prior to the incubation with the GST-fusion protein.
Article Title: Molecular functions of the double‐sided and inverted ubiquitin‐interacting motif found in Xenopus tropicalis cryptochrome 6
Article Snippet: Then, the sample was mixed with glutathione‐conjugated magnetic beads (MagneGST Glutathione Particles, Promega, V8611) and incubated with rotation for 1 h at 4°C. .. After unbound materials were washed out with the binding buffer, the precipitants were eluted with 50 mM glutathione (reduced form) in the binding buffer (pH 7.69) and analyzed by immunoblot analysis (Watari et al., ) using anti‐ubiquitin polyclonal antibody (Proteintech, 102021‐2‐AP) and anti‐GST monoclonal antibody (B‐14, Santa Cruz Biotechnology, sc‐138). .. In competition experiments, 2.2 μg of the polyubiquitin was mixed with 1 μg of synthetic polypeptide (XtCRY6DI‐UIM_WT, ADLETLGYETDLELAIALSLQEHNQLTD or XtCRY6DI‐UIM_SFL, ADLETLLEYGADLITAQESELLHNQLTD, Genscript) and incubated for 1 h at 4°C prior to the incubation with the GST‐fusion protein.
Protease Inhibitor:Article Title: Molecular functions of the double-sided and inverted ubiquitin-interacting motif found in Xenopus tropicalis cryptochrome 6.
Article Snippet: .. The purified GST-fusion protein (3 μg) was mixed with polyubiquitin (2.2 μg, K48Ub2–8, UBPBio, D1500) in binding buffer (25 mM HEPES[pH 7.2], 125 mM CH3COOK, 2.5 mM CH3COOMg, 5 mM EDTA, 0.5% TritonX-100, 1 mM dithiothreitol, 1 cOmplete protease inhibitor cocktail (Rosche, 4693116001) and incubated with rotation for 30 min at 4 C. Then, the sample was mixed with glutathione-conjugated magnetic beads (MagneGST Glutathione Particles, Promega, V8611) and incubated with rotation for 1 h at 4 C. After unbound materials were washed out with the binding buffer, the precipitants were eluted with 50 mM glutathione (reduced form) in the binding buffer (pH 7.69) and analyzed by immunoblot analysis (Watari et al., 2012) using anti-ubiquitin polyclonal antibody (Proteintech, 102021-2-AP) and anti-GST monoclonal antibody (B-14, Santa Cruz Biotechnology, sc-138). .. In competition experiments, 2.2 μg of the polyubiquitin was mixed with 1 μg of synthetic polypeptide (XtCRY6DI-UIM_WT, ADLETLGYETDLELAIALSLQEHNQLTD or XtCRY6DI-UIM_SFL, ADLETLLEYGADLITAQESELLHNQLTD, Genscript) and incubated for 1 h at 4 C prior to the incubation with the GST-fusion protein.
Incubation:Article Title: Molecular functions of the double-sided and inverted ubiquitin-interacting motif found in Xenopus tropicalis cryptochrome 6.
Article Snippet: .. The purified GST-fusion protein (3 μg) was mixed with polyubiquitin (2.2 μg, K48Ub2–8, UBPBio, D1500) in binding buffer (25 mM HEPES[pH 7.2], 125 mM CH3COOK, 2.5 mM CH3COOMg, 5 mM EDTA, 0.5% TritonX-100, 1 mM dithiothreitol, 1 cOmplete protease inhibitor cocktail (Rosche, 4693116001) and incubated with rotation for 30 min at 4 C. Then, the sample was mixed with glutathione-conjugated magnetic beads (MagneGST Glutathione Particles, Promega, V8611) and incubated with rotation for 1 h at 4 C. After unbound materials were washed out with the binding buffer, the precipitants were eluted with 50 mM glutathione (reduced form) in the binding buffer (pH 7.69) and analyzed by immunoblot analysis (Watari et al., 2012) using anti-ubiquitin polyclonal antibody (Proteintech, 102021-2-AP) and anti-GST monoclonal antibody (B-14, Santa Cruz Biotechnology, sc-138). .. In competition experiments, 2.2 μg of the polyubiquitin was mixed with 1 μg of synthetic polypeptide (XtCRY6DI-UIM_WT, ADLETLGYETDLELAIALSLQEHNQLTD or XtCRY6DI-UIM_SFL, ADLETLLEYGADLITAQESELLHNQLTD, Genscript) and incubated for 1 h at 4 C prior to the incubation with the GST-fusion protein.
Magnetic Beads:Article Title: Molecular functions of the double-sided and inverted ubiquitin-interacting motif found in Xenopus tropicalis cryptochrome 6.
Article Snippet: .. The purified GST-fusion protein (3 μg) was mixed with polyubiquitin (2.2 μg, K48Ub2–8, UBPBio, D1500) in binding buffer (25 mM HEPES[pH 7.2], 125 mM CH3COOK, 2.5 mM CH3COOMg, 5 mM EDTA, 0.5% TritonX-100, 1 mM dithiothreitol, 1 cOmplete protease inhibitor cocktail (Rosche, 4693116001) and incubated with rotation for 30 min at 4 C. Then, the sample was mixed with glutathione-conjugated magnetic beads (MagneGST Glutathione Particles, Promega, V8611) and incubated with rotation for 1 h at 4 C. After unbound materials were washed out with the binding buffer, the precipitants were eluted with 50 mM glutathione (reduced form) in the binding buffer (pH 7.69) and analyzed by immunoblot analysis (Watari et al., 2012) using anti-ubiquitin polyclonal antibody (Proteintech, 102021-2-AP) and anti-GST monoclonal antibody (B-14, Santa Cruz Biotechnology, sc-138). .. In competition experiments, 2.2 μg of the polyubiquitin was mixed with 1 μg of synthetic polypeptide (XtCRY6DI-UIM_WT, ADLETLGYETDLELAIALSLQEHNQLTD or XtCRY6DI-UIM_SFL, ADLETLLEYGADLITAQESELLHNQLTD, Genscript) and incubated for 1 h at 4 C prior to the incubation with the GST-fusion protein.
Western Blot:Article Title: Molecular functions of the double-sided and inverted ubiquitin-interacting motif found in Xenopus tropicalis cryptochrome 6.
Article Snippet: .. The purified GST-fusion protein (3 μg) was mixed with polyubiquitin (2.2 μg, K48Ub2–8, UBPBio, D1500) in binding buffer (25 mM HEPES[pH 7.2], 125 mM CH3COOK, 2.5 mM CH3COOMg, 5 mM EDTA, 0.5% TritonX-100, 1 mM dithiothreitol, 1 cOmplete protease inhibitor cocktail (Rosche, 4693116001) and incubated with rotation for 30 min at 4 C. Then, the sample was mixed with glutathione-conjugated magnetic beads (MagneGST Glutathione Particles, Promega, V8611) and incubated with rotation for 1 h at 4 C. After unbound materials were washed out with the binding buffer, the precipitants were eluted with 50 mM glutathione (reduced form) in the binding buffer (pH 7.69) and analyzed by immunoblot analysis (Watari et al., 2012) using anti-ubiquitin polyclonal antibody (Proteintech, 102021-2-AP) and anti-GST monoclonal antibody (B-14, Santa Cruz Biotechnology, sc-138). .. In competition experiments, 2.2 μg of the polyubiquitin was mixed with 1 μg of synthetic polypeptide (XtCRY6DI-UIM_WT, ADLETLGYETDLELAIALSLQEHNQLTD or XtCRY6DI-UIM_SFL, ADLETLLEYGADLITAQESELLHNQLTD, Genscript) and incubated for 1 h at 4 C prior to the incubation with the GST-fusion protein.
Article Title: Molecular functions of the double‐sided and inverted ubiquitin‐interacting motif found in Xenopus tropicalis cryptochrome 6
Article Snippet: Then, the sample was mixed with glutathione‐conjugated magnetic beads (MagneGST Glutathione Particles, Promega, V8611) and incubated with rotation for 1 h at 4°C. .. After unbound materials were washed out with the binding buffer, the precipitants were eluted with 50 mM glutathione (reduced form) in the binding buffer (pH 7.69) and analyzed by immunoblot analysis (Watari et al., ) using anti‐ubiquitin polyclonal antibody (Proteintech, 102021‐2‐AP) and anti‐GST monoclonal antibody (B‐14, Santa Cruz Biotechnology, sc‐138). .. In competition experiments, 2.2 μg of the polyubiquitin was mixed with 1 μg of synthetic polypeptide (XtCRY6DI‐UIM_WT, ADLETLGYETDLELAIALSLQEHNQLTD or XtCRY6DI‐UIM_SFL, ADLETLLEYGADLITAQESELLHNQLTD, Genscript) and incubated for 1 h at 4°C prior to the incubation with the GST‐fusion protein.
Infection:Article Title: PRRSV-2 impedes lysosomes from eliminating secondary infected bacteria.
Article Snippet: .. Anti-RhoA monoclonal antibody (#2117) was purchased from Cell Signaling Technology, Anti-Smurf1 (sc-100616) was purchased from Santa Cruz Biotechnology, anti-RhoA antibody (10749–1-AP, 66733–1-Ig), anti-ATG7 polyclonal antibody (10088–2-AP), anti-TOLLIP polyclonal antibody (11315–1-AP), anti-LAMP1 monoclonal antibody (CL647–65051), anti-ubiquitin Polyclonal antibody (80992–1-RR) were purchased from Proteintech, anti-tubulin monoclonal antibody (HC-101) was purchased from TransGen Biotech, anti-Flag antibody (20543–1-AP, CL594–66008), anti-Myc antibody (16286–1-AP, CL488–60003), anti-GFP polyclonal antibody (50430–2-AP), anti-HA antibody (51064–2-AP, CL647–81290) nsp5 and RhoA that interact with Smurf1 by Co-IP. (C) PAMs were infected with recombinant lentivirus expressing nsp5 or control lentivirus for 24 h, the cells were harvested to detect the interaction between nsp5 and endogenous RhoA, as well as endogenous Smurf1 by Co-IP. (D) HEK-293T cells were co-transfected with Flag-Smurf1, Myc-RhoA, and HA-nsp5 plasmids for 36 h, the cells were harvested to detect the subcellular localization of Smurf1, RhoA and nsp5. .. The co-localization of Smurf1, RhoA and nsp5 was analyzed using ImageJ, and the results were quantified using the Pearson’s correlation coefficient. n = 10 cells from three independent experiments. (E) HEK-293T cells were co-transfected with HA-nsp5, Flag- Smurf1 and MycRhoA plasmids for 36 h, the cells were harvested to detect the interaction between nsp5, RhoA and Smurf1 by ternary Co-IP. (F) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1 and Ub for 36 h, the cells were harvested to detect the ubiquitination of RhoA induced by Smurf1. (G) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1, nsp5 and Ub for 36 h, the cells were harvested to detect the effect of nsp5 on Smurf1-induced polyubiquitination of RhoA. (H) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1, nsp5 and K63-Ub for 36 h, the cells were harvested to detect the effect of nsp5 on Smurf1-induced polyubiquitination of RhoA. (I) HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and Smurf1 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (J) Smurf1+/+ and Smurf1-/- HEK-293T cells were co-transfected with the plasmids of RhoA and nsp5 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (K) Smurf1-/- HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and Smurf1 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (L) PAMs were transfected with siNC and siSmurf1 for 24 h, and then infected with recombinant lentivirus expressing nsp5 or control lentivirus for 24 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control.
Recombinant:Article Title: PRRSV-2 impedes lysosomes from eliminating secondary infected bacteria.
Article Snippet: .. Anti-RhoA monoclonal antibody (#2117) was purchased from Cell Signaling Technology, Anti-Smurf1 (sc-100616) was purchased from Santa Cruz Biotechnology, anti-RhoA antibody (10749–1-AP, 66733–1-Ig), anti-ATG7 polyclonal antibody (10088–2-AP), anti-TOLLIP polyclonal antibody (11315–1-AP), anti-LAMP1 monoclonal antibody (CL647–65051), anti-ubiquitin Polyclonal antibody (80992–1-RR) were purchased from Proteintech, anti-tubulin monoclonal antibody (HC-101) was purchased from TransGen Biotech, anti-Flag antibody (20543–1-AP, CL594–66008), anti-Myc antibody (16286–1-AP, CL488–60003), anti-GFP polyclonal antibody (50430–2-AP), anti-HA antibody (51064–2-AP, CL647–81290) nsp5 and RhoA that interact with Smurf1 by Co-IP. (C) PAMs were infected with recombinant lentivirus expressing nsp5 or control lentivirus for 24 h, the cells were harvested to detect the interaction between nsp5 and endogenous RhoA, as well as endogenous Smurf1 by Co-IP. (D) HEK-293T cells were co-transfected with Flag-Smurf1, Myc-RhoA, and HA-nsp5 plasmids for 36 h, the cells were harvested to detect the subcellular localization of Smurf1, RhoA and nsp5. .. The co-localization of Smurf1, RhoA and nsp5 was analyzed using ImageJ, and the results were quantified using the Pearson’s correlation coefficient. n = 10 cells from three independent experiments. (E) HEK-293T cells were co-transfected with HA-nsp5, Flag- Smurf1 and MycRhoA plasmids for 36 h, the cells were harvested to detect the interaction between nsp5, RhoA and Smurf1 by ternary Co-IP. (F) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1 and Ub for 36 h, the cells were harvested to detect the ubiquitination of RhoA induced by Smurf1. (G) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1, nsp5 and Ub for 36 h, the cells were harvested to detect the effect of nsp5 on Smurf1-induced polyubiquitination of RhoA. (H) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1, nsp5 and K63-Ub for 36 h, the cells were harvested to detect the effect of nsp5 on Smurf1-induced polyubiquitination of RhoA. (I) HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and Smurf1 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (J) Smurf1+/+ and Smurf1-/- HEK-293T cells were co-transfected with the plasmids of RhoA and nsp5 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (K) Smurf1-/- HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and Smurf1 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (L) PAMs were transfected with siNC and siSmurf1 for 24 h, and then infected with recombinant lentivirus expressing nsp5 or control lentivirus for 24 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control.
Expressing:Article Title: PRRSV-2 impedes lysosomes from eliminating secondary infected bacteria.
Article Snippet: .. Anti-RhoA monoclonal antibody (#2117) was purchased from Cell Signaling Technology, Anti-Smurf1 (sc-100616) was purchased from Santa Cruz Biotechnology, anti-RhoA antibody (10749–1-AP, 66733–1-Ig), anti-ATG7 polyclonal antibody (10088–2-AP), anti-TOLLIP polyclonal antibody (11315–1-AP), anti-LAMP1 monoclonal antibody (CL647–65051), anti-ubiquitin Polyclonal antibody (80992–1-RR) were purchased from Proteintech, anti-tubulin monoclonal antibody (HC-101) was purchased from TransGen Biotech, anti-Flag antibody (20543–1-AP, CL594–66008), anti-Myc antibody (16286–1-AP, CL488–60003), anti-GFP polyclonal antibody (50430–2-AP), anti-HA antibody (51064–2-AP, CL647–81290) nsp5 and RhoA that interact with Smurf1 by Co-IP. (C) PAMs were infected with recombinant lentivirus expressing nsp5 or control lentivirus for 24 h, the cells were harvested to detect the interaction between nsp5 and endogenous RhoA, as well as endogenous Smurf1 by Co-IP. (D) HEK-293T cells were co-transfected with Flag-Smurf1, Myc-RhoA, and HA-nsp5 plasmids for 36 h, the cells were harvested to detect the subcellular localization of Smurf1, RhoA and nsp5. .. The co-localization of Smurf1, RhoA and nsp5 was analyzed using ImageJ, and the results were quantified using the Pearson’s correlation coefficient. n = 10 cells from three independent experiments. (E) HEK-293T cells were co-transfected with HA-nsp5, Flag- Smurf1 and MycRhoA plasmids for 36 h, the cells were harvested to detect the interaction between nsp5, RhoA and Smurf1 by ternary Co-IP. (F) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1 and Ub for 36 h, the cells were harvested to detect the ubiquitination of RhoA induced by Smurf1. (G) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1, nsp5 and Ub for 36 h, the cells were harvested to detect the effect of nsp5 on Smurf1-induced polyubiquitination of RhoA. (H) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1, nsp5 and K63-Ub for 36 h, the cells were harvested to detect the effect of nsp5 on Smurf1-induced polyubiquitination of RhoA. (I) HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and Smurf1 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (J) Smurf1+/+ and Smurf1-/- HEK-293T cells were co-transfected with the plasmids of RhoA and nsp5 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (K) Smurf1-/- HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and Smurf1 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (L) PAMs were transfected with siNC and siSmurf1 for 24 h, and then infected with recombinant lentivirus expressing nsp5 or control lentivirus for 24 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control.
Control:Article Title: PRRSV-2 impedes lysosomes from eliminating secondary infected bacteria.
Article Snippet: .. Anti-RhoA monoclonal antibody (#2117) was purchased from Cell Signaling Technology, Anti-Smurf1 (sc-100616) was purchased from Santa Cruz Biotechnology, anti-RhoA antibody (10749–1-AP, 66733–1-Ig), anti-ATG7 polyclonal antibody (10088–2-AP), anti-TOLLIP polyclonal antibody (11315–1-AP), anti-LAMP1 monoclonal antibody (CL647–65051), anti-ubiquitin Polyclonal antibody (80992–1-RR) were purchased from Proteintech, anti-tubulin monoclonal antibody (HC-101) was purchased from TransGen Biotech, anti-Flag antibody (20543–1-AP, CL594–66008), anti-Myc antibody (16286–1-AP, CL488–60003), anti-GFP polyclonal antibody (50430–2-AP), anti-HA antibody (51064–2-AP, CL647–81290) nsp5 and RhoA that interact with Smurf1 by Co-IP. (C) PAMs were infected with recombinant lentivirus expressing nsp5 or control lentivirus for 24 h, the cells were harvested to detect the interaction between nsp5 and endogenous RhoA, as well as endogenous Smurf1 by Co-IP. (D) HEK-293T cells were co-transfected with Flag-Smurf1, Myc-RhoA, and HA-nsp5 plasmids for 36 h, the cells were harvested to detect the subcellular localization of Smurf1, RhoA and nsp5. .. The co-localization of Smurf1, RhoA and nsp5 was analyzed using ImageJ, and the results were quantified using the Pearson’s correlation coefficient. n = 10 cells from three independent experiments. (E) HEK-293T cells were co-transfected with HA-nsp5, Flag- Smurf1 and MycRhoA plasmids for 36 h, the cells were harvested to detect the interaction between nsp5, RhoA and Smurf1 by ternary Co-IP. (F) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1 and Ub for 36 h, the cells were harvested to detect the ubiquitination of RhoA induced by Smurf1. (G) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1, nsp5 and Ub for 36 h, the cells were harvested to detect the effect of nsp5 on Smurf1-induced polyubiquitination of RhoA. (H) HEK-293T cells were co-transfected with the plasmids of RhoA, Smurf1, nsp5 and K63-Ub for 36 h, the cells were harvested to detect the effect of nsp5 on Smurf1-induced polyubiquitination of RhoA. (I) HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and Smurf1 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (J) Smurf1+/+ and Smurf1-/- HEK-293T cells were co-transfected with the plasmids of RhoA and nsp5 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (K) Smurf1-/- HEK-293T cells were co-transfected with the plasmids of RhoA, nsp5 and Smurf1 for 36 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control. (L) PAMs were transfected with siNC and siSmurf1 for 24 h, and then infected with recombinant lentivirus expressing nsp5 or control lentivirus for 24 h, the cells were harvested to detect the RhoA, nsp5 and Smurf1 protein expression by Western blot, tubulin served as an internal control.
Transfection:Article Title: PRRSV-2 impedes lysosomes from eliminating secondary infected bacteria
Article Snippet: .. Anti-RhoA monoclonal antibody (#2117) was purchased from Cell Signaling Technology, Anti-Smurf1 (sc-100616) was purchased from Santa Cruz Biotechnology, anti-RhoA antibody (10749–1-AP, 66733–1-Ig), anti-ATG7 polyclonal antibody (10088–2-AP), anti-TOLLIP polyclonal antibody (11315–1-AP), anti-LAMP1 monoclonal antibody (CL647–65051), anti-ubiquitin Polyclonal antibody (80992–1-RR) were purchased from Proteintech, anti-tubulin monoclonal antibody (HC-101) was purchased from TransGen Biotech, anti-Flag antibody (20543–1-AP, CL594–66008), anti-Myc antibody (16286–1-AP, CL488–60003), anti-GFP polyclonal antibody (50430–2-AP), anti-HA antibody (51064–2-AP, CL647–81290) were purchased from Proteintech, anti-PRRSV-2 N monoclonal antibody was prepared by our laboratory; Lipofectamine RNAiMAX Transfection Reagent (13778150), Lipofectamine 3000 Transfection Reagent (L3000015) and LysoTracker Red DND-99 (L7528) were purchased from Thermo Fisher Scientific; CFDA (S1076) was purchased from Solarbio; YF 488-Phalloidin (YP0114L) was purchased from UElandy; Latrunculin A (HY-16929), 3 × Flag peptide (HY-P0319) were purchased from MedChemExpress; ELISA assay kits for porcine IL-1β (SEKP-0001) and IL-6 (SEKP-0004) were procured from Solarbio. .. Small interfering RNAs (siRNAs) against RhoA and negative control (NC) siRNA were designed and synthesized by Tsingke Biotechnology, and the sequences of the siRNAs used are listed in .
Enzyme-linked Immunosorbent Assay:Article Title: PRRSV-2 impedes lysosomes from eliminating secondary infected bacteria
Article Snippet: .. Anti-RhoA monoclonal antibody (#2117) was purchased from Cell Signaling Technology, Anti-Smurf1 (sc-100616) was purchased from Santa Cruz Biotechnology, anti-RhoA antibody (10749–1-AP, 66733–1-Ig), anti-ATG7 polyclonal antibody (10088–2-AP), anti-TOLLIP polyclonal antibody (11315–1-AP), anti-LAMP1 monoclonal antibody (CL647–65051), anti-ubiquitin Polyclonal antibody (80992–1-RR) were purchased from Proteintech, anti-tubulin monoclonal antibody (HC-101) was purchased from TransGen Biotech, anti-Flag antibody (20543–1-AP, CL594–66008), anti-Myc antibody (16286–1-AP, CL488–60003), anti-GFP polyclonal antibody (50430–2-AP), anti-HA antibody (51064–2-AP, CL647–81290) were purchased from Proteintech, anti-PRRSV-2 N monoclonal antibody was prepared by our laboratory; Lipofectamine RNAiMAX Transfection Reagent (13778150), Lipofectamine 3000 Transfection Reagent (L3000015) and LysoTracker Red DND-99 (L7528) were purchased from Thermo Fisher Scientific; CFDA (S1076) was purchased from Solarbio; YF 488-Phalloidin (YP0114L) was purchased from UElandy; Latrunculin A (HY-16929), 3 × Flag peptide (HY-P0319) were purchased from MedChemExpress; ELISA assay kits for porcine IL-1β (SEKP-0001) and IL-6 (SEKP-0004) were procured from Solarbio. .. Small interfering RNAs (siRNAs) against RhoA and negative control (NC) siRNA were designed and synthesized by Tsingke Biotechnology, and the sequences of the siRNAs used are listed in .
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