pcaggs ha vector  (New England Biolabs)

Journal: Molecular Microbiology

Article Title: Mycoplasma hyopneumoniae membrane protein Mhp271 interacts with host UPR protein GRP78 to facilitate infection

doi: 10.1111/mmi.14963

Figure Lengend Snippet: Mhp271 directly interacts with the host GRP78. (a and b) Mhp271‐Flag coimmunoprecipitated with GRP78‐Myc. HEK293T cells were transfected with Mhp271‐Flag or GRP78‐Myc, or the control plasmids pCAGGS‐Flag or pCAGGS‐Myc for 36 h. Cell lysates were collected for Co‐IP with beads conjugated with anti‐Flag antibody (a) and Myc antibody (b). (c) Far‐Western blotting analysis showed the binding of GRP78 with the Mhp271 protein. The proteins Mhp271‐MBP and PDH‐MBP (negative control) were transferred onto nitrocellulose (NC) membranes. Left: The NC membrane was incubated with anti‐MBP antibodies; middle: The NC membrane was first incubated with the GRP78‐GST protein and then with anti‐GRP78 antibodies; right: The NC membrane was first incubated with the GST protein and then with anti‐GST antibodies. (d) Colocalization of the Mhp271 and GRP78 proteins. HEK293T cells were transfected with GRP78‐Myc, Mhp271‐Flag, or both. Cells were subjected to indirect immunofluorescence to detect GRP78‐Myc (green) and Mhp271‐Flag (red) with mouse anti‐Myc and rabbit anti‐Flag antibodies, respectively. The position of the nucleus is indicated by DAPI (blue) staining in the merged image. Scale bars = 5 μm. Experiments were performed at least three times.

Article Snippet: The GRP78 gene was PCR amplified from PTECs and cloned into the pCAGGS‐HA vector (Clontech) with the restriction enzymes Kpn I and Xho I.

Techniques: Transfection, Co-Immunoprecipitation Assay, Far Western Blot, Binding Assay, Negative Control, Incubation, Immunofluorescence, Staining

Journal: Molecular Microbiology

Article Title: Mycoplasma hyopneumoniae membrane protein Mhp271 interacts with host UPR protein GRP78 to facilitate infection

doi: 10.1111/mmi.14963

Figure Lengend Snippet: Mhp271 plays an important role in M. hyopneumoniae infection to PTECs. (a and b) Mhp271 downregulated host PBD‐2 expression. PTECs were treated with PBS or the Mhp271 protein for 24 h (a) or transfected with pCAGGS‐flag, Mhp271c‐flag, or Mhp271c(ΔR1‐2) for 36 h (b), and PBD‐2 mRNA level was quantified by qPCR. (c–f) Mhp271 facilitated M. hyopneumoniae adherence to PTECs. PTECs were treated as described in panels A and B and then incubated with M. hyopneumoniae for 12 h. Cells were washed twice with PBS and harvested to analyze the number of M. hyopneumoniae cells adhered to PTECs by TaqMan qPCR (c and d), or cells were subjected to an M. hyopneumoniae P46 protein IFA with mouse anti‐ M. hyopneumoniae monoclonal antibody 3G11 (green) to detect M. hyopneumoniae adherence to PTECs. DAPI was used to stain cellular nuclei (blue) (e and f). Controls on uninfected cells with PBS or Mhp271 protein treatment or with pCAGGS‐Flag, Mhp271c‐Flag, or Mhp271c(ΔR1‐2) transfection are shown in Figure . (g) Murine serum against Mhp271 R1‐2 prevented Mhp271 from triggering host GRP78 degradation in PTECs, and 8 μg/ml Mhp271 protein was incubated with murine serum against Mhp271 R1‐2 (diluted 1:20, 1:50, 1:100, and 1:200) or negative murine serum (diluted 1:20) for 30 min and then added to PTECs. The GRP78 level in PTECs was determined by Western blotting. All assays were performed with three independent experiments, and values represent the means ± SD s. Significance was assessed by one‐way ANOVA with Dunnett's multiple comparison test relative to the control. * p < .05; ** p < .01.

Article Snippet: The GRP78 gene was PCR amplified from PTECs and cloned into the pCAGGS‐HA vector (Clontech) with the restriction enzymes Kpn I and Xho I.

Techniques: Infection, Expressing, Transfection, Incubation, Staining, Western Blot