anti-egfr neutralizing antibodies  (Millipore)

Journal: mBio

Article Title: Proteolytic Activity-Independent Activation of the Immune Response by Gingipains from Porphyromonas gingivalis

doi: 10.1128/mbio.03787-21

Figure Lengend Snippet: Blocking of receptors results in reduction of cytokine expression induced by inactive RgpA. TIGK cells were treated for 2 h with (A) inhibitors of the EGFR signaling pathway, including anti-EGFR neutralizing antibodies (5 μg/mL), cetuximab (10 μg/mL), and gefitinib (500 nM), (B) an inhibitor of the integrin α6β4 signaling pathway, anti-integrin β4 antibody, clone ASC-3 (10 μg/mL), and (C) an inhibitor of the Tfr1 signaling pathway, chlorazol black (ferristatin II) (20 μM). Cells were stimulated with inactive RgpA (2 nM) for 6 h, followed by evaluation of mRNA for IL-6 , TNF-α , and IL-1β expression by real-time PCR. Data are fold increase in expression compared to control levels, which were arbitrarily set at 1. Data are presented as means ± SEM from three independent assays. P values are noted as follows: # , P < 0.05, # # , P < 0.01, ## # , P < 0.001, and ### # , P < 0.0001, versus control; * * , P < 0.01, ** * , P < 0.001, and *** * , P < 0.0001, versus RgpA KYT -treated cells.

Article Snippet: TIGK cells (0.3 × 10 6 cells) were pretreated for 2 h with inhibitors of the EGFR signaling pathway, including anti-EGFR neutralizing antibodies (5 μg/mL; Sigma-Aldrich), cetuximab (10 μg/mL; InvivoGen), and gefitinib (500 nM; InvivoGen), an inhibitor of the integrin signaling pathway, anti-integrin β4 antibody (10 μg/mL; Sigma-Aldrich), and/or an inhibitor of the Tfr1 signaling pathway, chlorazol black (ferristatin II) (20 μM; Sigma-Aldrich).

Techniques: Blocking Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: mBio

Article Title: Proteolytic Activity-Independent Activation of the Immune Response by Gingipains from Porphyromonas gingivalis

doi: 10.1128/mbio.03787-21

Figure Lengend Snippet: Inactive gingipain RgpA activates immune responses via the EGFR-PI3K-AKT pathway. (A) Confocal laser scanning microscopy presenting colocalization of RgpA with EGFR. Gingival keratinocytes were stimulated with inactivated RgpA (2 nM) for 1 h. Slides were stained against RgpA (Alexa-Fluor 488 [green]), EGFR (Alexa-Fluor 647 [red]), and cell nuclei (Hoechst 33342, blue), and slides were analyzed at ×100 magnification. The Mander’s overlap coefficient (MOC) was measured from 27 images. (B) Representative Western Blot analysis of phosphorylation of the EGFR receptor after 30 min of stimulation with active and inactive RgpA (2 nM). (C) Ratio of pEGFR(Y1173) to total EGFR. (D) Gingival keratinocytes were stimulated for 3 h with PI3K inhibitor LY294002 (10 μg/mL), and then active or inactivated gingipain RgpA (2 nM) was added. After 6 h of stimulation, expression of IL-6 mRNA was evaluated by real-time PCR. (E, G) Representative Western blot analysis of phosphorylation of AKT after 1 h of stimulation with (E) active and inactivated RgpA (2 nM) or (G) strain W83 in the presence or absence of KYT inhibitors (KYT-1 and KYT-36, each at 1 μM). (F, H) Relative expression of pAKT(T308) to GAPDH. Data are presented as means ± SEM from three independent assays. P values are noted as follows: #, P < 0.05, and ### # , P < 0.0001 versus control; * , P < 0.05, * * , P < 0.01, and *** * , P < 0.0001, versus RgpA- or bacterium-treated cells.

Article Snippet: TIGK cells (0.3 × 10 6 cells) were pretreated for 2 h with inhibitors of the EGFR signaling pathway, including anti-EGFR neutralizing antibodies (5 μg/mL; Sigma-Aldrich), cetuximab (10 μg/mL; InvivoGen), and gefitinib (500 nM; InvivoGen), an inhibitor of the integrin signaling pathway, anti-integrin β4 antibody (10 μg/mL; Sigma-Aldrich), and/or an inhibitor of the Tfr1 signaling pathway, chlorazol black (ferristatin II) (20 μM; Sigma-Aldrich).

Techniques: Confocal Laser Scanning Microscopy, Staining, Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Exposure to mold proteases stimulates mucin production in airway epithelial cells through Ras/Raf1/ERK signal pathway

doi: 10.1371/journal.pone.0231990

Figure Lengend Snippet: The cells were pre-treated with EGFR inhibitors, BIBX1382 (5 μM), AG1478 (2 μM) for 1 hr, and then treated with 7.5 μg/ml of AFE for 6 hrs. (A) MUC5AC and (B) MUC5B were quantified by Real-Time PCR. (C-D) The cells were pre-treated with 1 μg/ml of neutralizing anti-EGFR antibody and then AFE stimulation for 6 hrs. (C) MUC5AC and (D) MUC5B were measured. (E) The cells were stimulated with EGFR (positive control) and 7.5 μg/ml of AFE for 1, 3, 6 hrs and then probed with pEGFR and β- ACTIN. (F) The cells were stimulated with 7.5 μg/ml of AFE for 1, 3, 6 hr, and then immuneprecipitated with EGFR antibody. pEGFR was blotted with phosphotysine antibody (pTyr). Total EGFR was also blotted for the input control. (G) The cells were stimulated with 50 ng/ml of EGF for 1 or 6 hrs, and then probed with pEGFR and β- ACTIN. (H) The cells were stimulated with 50 ng/ml of EGF for 6 hrs. and then harvested for Real-time PCR analysis for MUC5AC and MUC5B. #, *: P < 0.05.

Article Snippet: AG1478 (Sigma, St. Louis, MO), BIBX 1382 (Sigma, St. Louis, MO), neutralizing anti-EGFR antibody (Calbiochem, La Jolla, CA), Raf-1 inhibitor (Sigma, St. Louis, MO) and sorafenib (LC laboratories, Woburn, MA), U0126 (1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio] butadiene) (Sigma, St. Louis, MO).

Techniques: Real-time Polymerase Chain Reaction, Positive Control