influenza a h1n1 m2 antibody (14c2) - (a/wsn/1933)  (Bio-Techne corporation)

Journal: The FASEB Journal

Article Title: Influenza virus M2 targets cystic fibrosis transmembrane conductance regulator for lysosomal degradation during viral infection

doi: 10.1096/fj.14-268755

Figure Lengend Snippet: Infection of polarized airway epithelial cell monolayers with influenza virus reduces CFTR activity and protein levels. A) Transepithelial currents recorded from influenza Udorn–infected NHBE cell monolayers at 18 hours postinfection. Representative tracing of mock-infected (solid line) and influenza Udorn–infected cells (dashed line) with addition of inhibitors and activators as indicated. Forskolin (Forsk)-stimulated (Stim) (10 µM), GlyH-101 (GlyH)-sensitive (Sens) (50 µM), amiloride (Amil)-sensitive currents in Udorn-infected cells were plotted relative to currents recorded from mock-infected cells. Transepithelial resistances were also plotted relative to mock-infected cells (n = 9 mock, n = 6 Udorn). B) Transepithelial currents recorded from influenza PR8-infected MNE cell monolayers at 24 hours postinfection (MOI = 3). Representative trace of mock-infected (solid line) and influenza PR8-infected cells (dashed line) with addition of inhibitors and activators as indicated. Forskolin-stimulated (10 µM), GlyH-101-sensitive (50 µM), and amiloride-sensitive currents are plotted relative to mock. Transepithelial resistances are plotted on the right, relative to mock (n = 9 mock, n = 5; PR8, forsk stim PR8; n = 4). C) CFTR, M1, and M2 protein expression in mock-infected and influenza Udorn-infected NHBE cells (MOI = 3), 18 hours postinfection. CFTR levels in influenza-infected cells (normalized to β-actin) were plotted relative to CFTR expression in mock-infected NHBE cells (n = 6). All values are means ± sd. *P < 0.05 indicates significant difference from mock-infected control by Student’s t test, or by ANOVA for 3 or more groups.

Article Snippet: We also used M2 antibody (14C2; Novus Biologicals, Littleton, CO, USA) and Influenza M1 antibody (GA2B; Abcam, Cambridge, MA, USA).

Techniques: Infection, Virus, Activity Assay, Expressing

Journal: The FASEB Journal

Article Title: Influenza virus M2 targets cystic fibrosis transmembrane conductance regulator for lysosomal degradation during viral infection

doi: 10.1096/fj.14-268755

Figure Lengend Snippet: Reduced CFTR expression in HEK CFTRwt cells following influenza (Udorn) virus infection. A) Time course studies in HEK CFTRwt cells following influenza Udorn infection (MOI = 1). CFTR, M1, and M2 protein levels were tested by Western blotting. B-actin was labeled as loading control. B) Western blot of biotinylated plasma membrane CFTR in uninfected and PR8-infected HEK CFTRwt cells 24 hours postinfection (top). Plasma membrane CFTR levels following PR8 infection were plotted relative to surface CFTR levels in uninfected (UN) cells (n = 5). C) Bright-field + GFP fluorescent images of uninfected and NS1-GFP PR8-infected HEK CFTRwt cells at MOI = 1, 24 hours postinfection. D) Western blots of total CFTR and M1 expression in uninfected and NS1-GFP PR8-infected HEK CFTRwt cells. *P < 0.05 as compared to uninfected cells.

Article Snippet: We also used M2 antibody (14C2; Novus Biologicals, Littleton, CO, USA) and Influenza M1 antibody (GA2B; Abcam, Cambridge, MA, USA).

Techniques: Expressing, Virus, Infection, Western Blot, Labeling, Membrane

Journal: The FASEB Journal

Article Title: Influenza virus M2 targets cystic fibrosis transmembrane conductance regulator for lysosomal degradation during viral infection

doi: 10.1096/fj.14-268755

Figure Lengend Snippet: Efficient knockdown of influenza M2 expression by DsiRNA in influenza infected HEK CFTRwt cells reduces M2 activity. A) HEK-293 CFTRwt cells were untransfected (Untrans), DsiScram-transfected (DsiScram), or transfected with DsiRNA against 2 distinct M2 mRNA targets (DsiM2-1, DsiM2-3). Cells were then infected with influenza Udorn virus 72 hours post-transfection. Twenty-four hours postinfection total M1 and M2 expression was measured. B) Experimental scheme of DsiRNA transfection/NS1-GFP infection procedure for whole-cell patch clamp experiments. At 0 hours cells were seeded onto 6 well plates. At 24 hours postseeding, cells were transfected with DsiRNA. Transfected cells were trypsinized and seeded onto coverslips 24 hours post-transfection. Twenty-four hours later, cells were infected with NS1-GFP PR8 at MOI = 0.4. After another 24 hours, we measured whole-cell currents. C) (Left panel) Plots of whole-cell patch clamp recorded current-voltage (I-V) relationships of pH-induced currents. Whole-cell currents were measured by subtracting basal currents at pH 7.6 from currents after perfusion with a pH 5.5 buffer. (Right panel) Quantification of pH-induced currents at −80 mV in DsiRNA-transfected HEK CFTRwt cells (uninfected: light gray bars; NS1-GFP PR8–infected: white bars) (n = 6, 6, 21, 15). ΔI pH 5.5 (pA) = difference in currents between 5.5 and 7.4. All values are means ± 1 sd. *P < 0.05 indicates significant difference from untransfected control, #P < 0.05 = significantly different from DsiScram-transfected, NS1-GFP PR8-infected cells by Student’s t test, or by ANOVA for 3 or more groups.

Article Snippet: We also used M2 antibody (14C2; Novus Biologicals, Littleton, CO, USA) and Influenza M1 antibody (GA2B; Abcam, Cambridge, MA, USA).

Techniques: Expressing, Infection, Activity Assay, Transfection, Virus, Patch Clamp

Journal: The FASEB Journal

Article Title: Influenza virus M2 targets cystic fibrosis transmembrane conductance regulator for lysosomal degradation during viral infection

doi: 10.1096/fj.14-268755

Figure Lengend Snippet: Measurement of apoptosis/necrosis in influenza NS1-GFP PR8-infected HEK CFTRwt cells. HEK CFTRwt cells were infected with influenza NS1-GFP at MOI = 2. At 24 hours postinfection, cells were trypsinized, stained, and analyzed by flow cytometry. Sorting was gated for annexin V to detect early apoptosis and Necrosis Detection Reagent to detect necrosis. A) Representative sortings of unstained cells, staurosporine-induced apoptotic cells (15 µM, 4 hours, positive control), uninfected (Un) cells, and NS1-GFP PR8-infected cells. [7-AAD = necrotic, Enzo-Gold (annexin 5) = apoptotic; red cells = GFP-; green = GFP+]. B) Quantification of apoptosis/necrosis in staurosporine-treated, uninfected and NS1-GFP-infected cells without (Dsi Scr) and with (Dsi M2) M2 knockdown. The total cell population was analyzed. Apoptotic = white bar; apoptotic/necrotic = light gray bar; necrotic = dark gray bar (n = 2, 3, 3, 3, 6, 6, 6; data from 2 experiments). C) Analysis of GFP-positive (virus-infected) cells only for apoptosis and necrosis without (Dsi Scr) and with (Dsi M2) M2 knockdown (n = 6,6,6). NS, not significant. All values are means ± 1 sd. *P < 0.05 = significantly different from uninfected control by Student’s t test, or by ANOVA for 3 or more groups.

Article Snippet: We also used M2 antibody (14C2; Novus Biologicals, Littleton, CO, USA) and Influenza M1 antibody (GA2B; Abcam, Cambridge, MA, USA).

Techniques: Infection, Staining, Flow Cytometry, Positive Control, Virus

Journal: The FASEB Journal

Article Title: Influenza virus M2 targets cystic fibrosis transmembrane conductance regulator for lysosomal degradation during viral infection

doi: 10.1096/fj.14-268755

Figure Lengend Snippet: No reduction in CFTR expression and activity following M2 knockdown with DsiRNA in HEK CFTRwt cells. A) HEK CFTRwt cells were transfected with plasmids expressing M2-GFP or GFP alone. At 48 hours post-transfection, CFTR expression levels were measured in whole-cell lysates (WCL) and at the plasma membrane (PM). Representative gels are shown on left. CFTR levels in WCL and at the plasma membrane are plotted on right. GFP: gray bar; M2-GFP: white bar. WCL (n = 9, 9). PM (n = 13, 11). B) HEK CFTRwt cells were transfected and infected as described in Figure 4B. Representative images of Western blots (left) of total CFTR band C, M1, and M2 expression in uninfected/untransfected, transfected with DsiScram (Scram), or transfected with DsiM2 (M2) versus PR8-infected/untransfected (none), transfected with DsiScram (Scram), or transfected with DsiM2 (M2). Quantification of CFTR band C expression levels (right) in DsiScram v. DsiM2-transfected cells infected with NS1-GFP (n = 10, 10, 9, 9). C) Representative tracing of forskolin (Forsk) + IBMX-induced, GlyH-101 + PPQ-inhibited currents in uninfected (black line) and NS1-GFP PR8 -infected (light gray line) cells (left). Summary of CFTR inhibitor-sensitive conductances in uninfected or NS1-GFP PR8-infected cells with or without M2 knockdown (right, uninfected: light gray bars; NS1-GFP PR8-infected: white bars; n = 9, 10, 7, 7, 13, 16). All values are means ± 1 sd. *P < 0.05 = significantly different from uninfected, untransfected control, #P < 0.05 = significantly different from DsiScram-transfected, NS1-GFP PR8-infected cells by Student’s t test, or by ANOVA for 3 or more groups. D) Single-channel patch clamp analysis of HEK-293 CFTRwt in control, NS1-GFP-PR8 virus -infected, and NS1-GFP-PR8 virus-infected plus M2 DsiRNA-transfected cells. Forskolin induced multiple, 7 pS channel openings, consistent with CFTR function in control cells (top). V NS1-GFP-PR8 virus infection significantly reduced CFTR activity (middle). Knockdown of M2 following NS1-GFP-PR8 virus infection resulted in the recovery of CFTR activity (bottom). Representative tracings are shown on right. Summary of single-channel experiments are shown on right with calculated open probabilities. Mean values ± sd for NPo (number of measurements) for these conditions were as follows: control = 1 ± 0.1.4 (n = 11); virus = 0.06 ± 0.01455 (n = 9) (P < 0.01 compared with control); DsiRNA+ Virus = 0.63 ± 0.1 (n = 10); (P < 0.01 compared with virus alone; by ANOVA followed by Tukey-Kramer multiple comparison test).

Article Snippet: We also used M2 antibody (14C2; Novus Biologicals, Littleton, CO, USA) and Influenza M1 antibody (GA2B; Abcam, Cambridge, MA, USA).

Techniques: Expressing, Activity Assay, Transfection, Membrane, Infection, Western Blot, Patch Clamp, Virus, Comparison

Journal: The FASEB Journal

Article Title: Influenza virus M2 targets cystic fibrosis transmembrane conductance regulator for lysosomal degradation during viral infection

doi: 10.1096/fj.14-268755

Figure Lengend Snippet: Inhibition of M2 activity with amantadine (AMA) in influenza Udorn-infected cells eliminates the reduction in CFTR levels. A) Representative Western blots of CFTR M1 and M2 levels in HEK CFTRwt cells infected with the amantadine-sensitive Udorn virus (top) or the amantadine-insensitive, NS1-GFP PR8 virus (lower gel image). AMA was added at indicated concentrations 2 hours postinfection. B) Summary of M2 inhibition studies on CFTR expression levels following Udorn or NS1-GFP-PR8 virus infections (n = 4, 2, 2, 5, 6, 4, 3, 3, 3). All values are means ± 1 sd. *P < 0.05 = significantly different from uninfected, untransfected control, #P < 0.05 indicates significant difference from DsiScram-transfected, NS1-GFP PR8–infected cells by Student’s t test, or by ANOVA for 3 or more groups.

Article Snippet: We also used M2 antibody (14C2; Novus Biologicals, Littleton, CO, USA) and Influenza M1 antibody (GA2B; Abcam, Cambridge, MA, USA).

Techniques: Inhibition, Activity Assay, Infection, Western Blot, Virus, Expressing, Transfection

Journal: The FASEB Journal

Article Title: Influenza virus M2 targets cystic fibrosis transmembrane conductance regulator for lysosomal degradation during viral infection

doi: 10.1096/fj.14-268755

Figure Lengend Snippet: Influenza virus infection directs CFTR to the lysosome. A) Effects of lysosome and proteasome inhibitors on CFTR levels following Udorn infection. HEK-293 CFTRwt cells were uninfected, or infected with influenza Udorn at MOI1. To inhibit the lysosome, BafA1was added at the concentrations indicated 6 hours postinfection. Levels of CFTR, M1, M2, and β-actin were measured via Western blot (WB) (top panel). The effects of proteasome inhibition were also tested in uninfected and Udorn-infected cells (middle panel). Lactacystin was added 18 hours postinfection at indicated concentrations to inhibit the proteasome for 6 hours. β-actin levels were determined as sample loading control. CFTR band C levels in uninfected cells and postinfection with influenza Udorn were plotted relative to β-actin in the absence or presence of either lactacystin or BafA1 treatment (n = 18, 13, 5, 17, 12, 5). All values are means ± sd. *P < 0.05, indicates significant difference from uninfected. #P < 0.05 indicates significant difference from NS1-GFP PR8–infected cells by Student’s t test, or by ANOVA for 3 or more groups. B) BafA1 eliminates influenza Udorn-associated reduction in CFTR levels and increases the levels of K-63-linked polyubiquitinated CFTR. Uninfected (Un) or Udorn-infected (MOI = 1) HEK-293 CFTRwt cells were tested in the absence (−) or presence (+) of Bafilomycin A1 (BafA1). BafA1 was added 6 hours postinfection. CFTR was immunoprecipitated (IP) and probed for K-63-linked polyubiquitination via Western blotting. Representative gels are shown on left. K-63-linked polyubiquitination levels were plotted relative to total immunoprecipitated CFTR levels in control or Udorn-infected cells (right). C) K-48-linked ubiquitination of CFTR. Cells were treated identical to B. CFTR was immunoprecipitated and probed for K-48-linked polyubiquitination via Western blotting. β-actin served as loading control. K-4848-linked polyubiquitination levels were plotted relative to total immunoprecipitated CFTR levels. D) Proteasome inhibition with lactacystin does not recover CFTR levels in Udorn-infected cells. Uninfected (Un) or Udorn-infected (Ud) HEK-293 CFTRwt cells were tested in the absence (−) or presence (+) of lactacystin. Lactacystin was added 18 hours postinfection to inhibit proteasomal degradation. CFTR was immunoprecipitated and probed for K-48-linked polyubiquitination via Western blotting. Representative gels are shown on left. K-48-linked ubiquitination levels were plotted relative to total CFTR levels in uninfected or Udorn-infected cells. All values are means ± n = 3. *P < 0.05 (Student’s t test). β-actin was detected in (A–D) as loading control.

Article Snippet: We also used M2 antibody (14C2; Novus Biologicals, Littleton, CO, USA) and Influenza M1 antibody (GA2B; Abcam, Cambridge, MA, USA).

Techniques: Virus, Infection, Western Blot, Inhibition, Immunoprecipitation