rabbit polyclonal ifitm3 antibody  (Proteintech)

Journal: Microorganisms

Article Title: Interferon-Induced Transmembrane Protein 3 (IFITM3) Restricts PRRSV Replication via Post-Entry Mechanisms

doi: 10.3390/microorganisms13081737

Figure Lengend Snippet: The over-expression of IFITM3 reduces PRRSV replication. ( a ) Western blotting analysis showing the expression of exogenous IFITM3 in cells transfected with a plasmid containing IFITM3 (I), but not in cells transfected with vector control (V). ( b ) Virus titers in the supernatants of cells transfected with vector control or IFITM3 containing plasmid at 24 h after virus infection. An average of a 5.4-fold decrease was observed in IFITM3-transfected cells compared to vector controls. ( c ) CCK-8 cytotoxicity assay was used to compare the difference in cell viability between QCXIP vector control and IFITM3-HA over-expressing MARC-145 cells. A p -value < 0.05 was considered statistically significant.

Article Snippet: The following primary antibodies were used: anti-HA mouse monoclonal 1o Ab (6E2) (Cell Signaling Technology, catalog# 2367) at 1:1000 dilution, anti-HA mouse monoclonal antibody (HA7) (Sigma Aldrich, catalog# H3663, Saint Louis, MO, USA) at 1:5000 dilution, anti-PRRSV SR-30 1o monoclonal Ab (provided by Dr. Eric Nelson) at 1:300 dilution, anti-IFITM3 rabbit polyclonal antibody (Proteintech, catalog# 11714-1-AP, Rosemont, IL, USA) at 1:1000 dilution, anti-p-Akt (S743, Cell Signaling Technology, catalog# 9271S) at 1:1000 dilution, anti-LC3B antibody (Sigma Aldrich, catalog# L7542) at 1:2000 dilution, and mouse monoclonal anti-beta actin Ab (Sigma Aldrich, catalog# A2228) at 1:5000 dilution.

Techniques: Over Expression, Western Blot, Expressing, Transfection, Plasmid Preparation, Control, Virus, Infection, CCK-8 Assay, Cytotoxicity Assay

Journal: Microorganisms

Article Title: Interferon-Induced Transmembrane Protein 3 (IFITM3) Restricts PRRSV Replication via Post-Entry Mechanisms

doi: 10.3390/microorganisms13081737

Figure Lengend Snippet: The silencing of IFITM3 slightly enhances PRRSV replication. ( a ) qRT-PCR showing the silencing of IFITM3. The averages and standard deviations of three replicates are shown. ( b ) qRT-PCR showing the viral RNA copies in control silencing and IFITM3 silencing RNA-transfected cells. The averages and standard deviations of three replicates are shown. ( c ) A slightly enhanced TCID 50 virus titer (1.22-fold increase) in IFITM3 silencing RNA-transfected cells compared to control silencing RNA-transfected cells was observed. The averages and standard deviations of three replicates are shown. ( d ) The difference in cell viability was compared between the negative control siRNA and IFITM3 siRNA-transfected MARC-145 cells. The graph shows the mean absorbance values read at 450 nm between the two groups. A p -value < 0.05 was considered significant.

Article Snippet: The following primary antibodies were used: anti-HA mouse monoclonal 1o Ab (6E2) (Cell Signaling Technology, catalog# 2367) at 1:1000 dilution, anti-HA mouse monoclonal antibody (HA7) (Sigma Aldrich, catalog# H3663, Saint Louis, MO, USA) at 1:5000 dilution, anti-PRRSV SR-30 1o monoclonal Ab (provided by Dr. Eric Nelson) at 1:300 dilution, anti-IFITM3 rabbit polyclonal antibody (Proteintech, catalog# 11714-1-AP, Rosemont, IL, USA) at 1:1000 dilution, anti-p-Akt (S743, Cell Signaling Technology, catalog# 9271S) at 1:1000 dilution, anti-LC3B antibody (Sigma Aldrich, catalog# L7542) at 1:2000 dilution, and mouse monoclonal anti-beta actin Ab (Sigma Aldrich, catalog# A2228) at 1:5000 dilution.

Techniques: Quantitative RT-PCR, Control, Transfection, Virus, Negative Control

Journal: Microorganisms

Article Title: Interferon-Induced Transmembrane Protein 3 (IFITM3) Restricts PRRSV Replication via Post-Entry Mechanisms

doi: 10.3390/microorganisms13081737

Figure Lengend Snippet: The IFITM3 expression level is inversely correlated to PRRSV replication efficiency. ( a ) IFN-alpha induces IFITM3 expression. Relative fold changes in IFITM3 and Mx1 gene expression in PRRSV 23,983-infected groups when compared to mock-treated groups are shown. More abundant Mx1 as compared to IFITM3 mRNA is induced by IFN-alpha. Representative data show the average and standard deviation of three replicates. ( b ) IFN-alpha inhibits PRRSV infection in a dose-dependent manner. Real-time RT-PCR shows the relative fold changes in IFITM3 and PRRSV N mRNA gene expression in IFN-alpha-treated groups at different concentrations when compared to non-treated control groups. Representative data show the average of two replicates. ( c ) The over-expression of IFITM3 results in 6.2-fold increase in the total IFITM3 protein level compared to the endogenous IFITM3 expression level. ( d ) No virus titer was detected in cells over-expressing IFITM3 while 1.9 × 10 4 TCID 50 titer was detected in cells only expressing the endogenous level of IFITM3.

Article Snippet: The following primary antibodies were used: anti-HA mouse monoclonal 1o Ab (6E2) (Cell Signaling Technology, catalog# 2367) at 1:1000 dilution, anti-HA mouse monoclonal antibody (HA7) (Sigma Aldrich, catalog# H3663, Saint Louis, MO, USA) at 1:5000 dilution, anti-PRRSV SR-30 1o monoclonal Ab (provided by Dr. Eric Nelson) at 1:300 dilution, anti-IFITM3 rabbit polyclonal antibody (Proteintech, catalog# 11714-1-AP, Rosemont, IL, USA) at 1:1000 dilution, anti-p-Akt (S743, Cell Signaling Technology, catalog# 9271S) at 1:1000 dilution, anti-LC3B antibody (Sigma Aldrich, catalog# L7542) at 1:2000 dilution, and mouse monoclonal anti-beta actin Ab (Sigma Aldrich, catalog# A2228) at 1:5000 dilution.

Techniques: Expressing, Gene Expression, Infection, Standard Deviation, Quantitative RT-PCR, Control, Over Expression, Virus

Journal: Microorganisms

Article Title: Interferon-Induced Transmembrane Protein 3 (IFITM3) Restricts PRRSV Replication via Post-Entry Mechanisms

doi: 10.3390/microorganisms13081737

Figure Lengend Snippet: Amphotericin B treatment only partially restores PRRSV replication in cells over-expressing IFITM3. ( a ) Immunofluorescence staining of virus-infected cells in the presence or absence of amphotericin B treatment. Images were captured at 10× magnification. ( b ) Flow cytometry analysis of virus-infected cells in the presence or absence of Amphotericin B. * indicates p < 0.05 when compared to the pQCXIP + PRRSV group.

Article Snippet: The following primary antibodies were used: anti-HA mouse monoclonal 1o Ab (6E2) (Cell Signaling Technology, catalog# 2367) at 1:1000 dilution, anti-HA mouse monoclonal antibody (HA7) (Sigma Aldrich, catalog# H3663, Saint Louis, MO, USA) at 1:5000 dilution, anti-PRRSV SR-30 1o monoclonal Ab (provided by Dr. Eric Nelson) at 1:300 dilution, anti-IFITM3 rabbit polyclonal antibody (Proteintech, catalog# 11714-1-AP, Rosemont, IL, USA) at 1:1000 dilution, anti-p-Akt (S743, Cell Signaling Technology, catalog# 9271S) at 1:1000 dilution, anti-LC3B antibody (Sigma Aldrich, catalog# L7542) at 1:2000 dilution, and mouse monoclonal anti-beta actin Ab (Sigma Aldrich, catalog# A2228) at 1:5000 dilution.

Techniques: Expressing, Immunofluorescence, Staining, Virus, Infection, Flow Cytometry

Journal: Microorganisms

Article Title: Interferon-Induced Transmembrane Protein 3 (IFITM3) Restricts PRRSV Replication via Post-Entry Mechanisms

doi: 10.3390/microorganisms13081737

Figure Lengend Snippet: Over-expression of IFITM3 does not significantly impact virus entry. ( a ) Cells over-expressing exogenous IFITM3 contain PRRSV at 3 and 24 hpi. The colocalization of PRRSV with over-expressed IFITM3 was observed (yellow arrows). Red: IFITM3; green: PRRSV N; blue: DAPI. The images were taken at 40× magnification. ( b ) The percentage of PRRSV-positive cells was significantly lower ( p < 0.0001) in IFITM3-transfected cells at 24 hpi than at 3 hpi. Quantification was performed by counting PRRSV-positive cells and IFITM3-HA-expressing cells obtained from five different microscopic fields. * indicates p < 0.0001.

Article Snippet: The following primary antibodies were used: anti-HA mouse monoclonal 1o Ab (6E2) (Cell Signaling Technology, catalog# 2367) at 1:1000 dilution, anti-HA mouse monoclonal antibody (HA7) (Sigma Aldrich, catalog# H3663, Saint Louis, MO, USA) at 1:5000 dilution, anti-PRRSV SR-30 1o monoclonal Ab (provided by Dr. Eric Nelson) at 1:300 dilution, anti-IFITM3 rabbit polyclonal antibody (Proteintech, catalog# 11714-1-AP, Rosemont, IL, USA) at 1:1000 dilution, anti-p-Akt (S743, Cell Signaling Technology, catalog# 9271S) at 1:1000 dilution, anti-LC3B antibody (Sigma Aldrich, catalog# L7542) at 1:2000 dilution, and mouse monoclonal anti-beta actin Ab (Sigma Aldrich, catalog# A2228) at 1:5000 dilution.

Techniques: Over Expression, Virus, Expressing, Transfection

Journal: Microorganisms

Article Title: Interferon-Induced Transmembrane Protein 3 (IFITM3) Restricts PRRSV Replication via Post-Entry Mechanisms

doi: 10.3390/microorganisms13081737

Figure Lengend Snippet: The over-expression of IFITM3 reduces the level of phosphorylated Akt by 50% compared to the vector control. A representative Western blot image shows the expression level of p-Akt. The averages and standard deviations of three replicates are shown in the graph. A significant decrease in the level of p-Akt was observed in IFITM3-over-expressing and PRRSV-infected cells compared to vector-transfected and PRRSV-infected cells ( p < 0.05).

Article Snippet: The following primary antibodies were used: anti-HA mouse monoclonal 1o Ab (6E2) (Cell Signaling Technology, catalog# 2367) at 1:1000 dilution, anti-HA mouse monoclonal antibody (HA7) (Sigma Aldrich, catalog# H3663, Saint Louis, MO, USA) at 1:5000 dilution, anti-PRRSV SR-30 1o monoclonal Ab (provided by Dr. Eric Nelson) at 1:300 dilution, anti-IFITM3 rabbit polyclonal antibody (Proteintech, catalog# 11714-1-AP, Rosemont, IL, USA) at 1:1000 dilution, anti-p-Akt (S743, Cell Signaling Technology, catalog# 9271S) at 1:1000 dilution, anti-LC3B antibody (Sigma Aldrich, catalog# L7542) at 1:2000 dilution, and mouse monoclonal anti-beta actin Ab (Sigma Aldrich, catalog# A2228) at 1:5000 dilution.

Techniques: Over Expression, Plasmid Preparation, Control, Western Blot, Expressing, Infection, Transfection

Journal: Microorganisms

Article Title: Interferon-Induced Transmembrane Protein 3 (IFITM3) Restricts PRRSV Replication via Post-Entry Mechanisms

doi: 10.3390/microorganisms13081737

Figure Lengend Snippet: The over-expression of IFITM3 increases the ratio of LC3-II/LC3-I by 128% compared to vector control. A representative Western blot image shows the expression level of LC3-I and LC3-II. The averages and standard deviations of three replicates are shown in the graph. * Indicates significant differences compared to mock ( p < 0.05).

Article Snippet: The following primary antibodies were used: anti-HA mouse monoclonal 1o Ab (6E2) (Cell Signaling Technology, catalog# 2367) at 1:1000 dilution, anti-HA mouse monoclonal antibody (HA7) (Sigma Aldrich, catalog# H3663, Saint Louis, MO, USA) at 1:5000 dilution, anti-PRRSV SR-30 1o monoclonal Ab (provided by Dr. Eric Nelson) at 1:300 dilution, anti-IFITM3 rabbit polyclonal antibody (Proteintech, catalog# 11714-1-AP, Rosemont, IL, USA) at 1:1000 dilution, anti-p-Akt (S743, Cell Signaling Technology, catalog# 9271S) at 1:1000 dilution, anti-LC3B antibody (Sigma Aldrich, catalog# L7542) at 1:2000 dilution, and mouse monoclonal anti-beta actin Ab (Sigma Aldrich, catalog# A2228) at 1:5000 dilution.

Techniques: Over Expression, Plasmid Preparation, Control, Western Blot, Expressing