Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Molecular and Cellular Biology

Article Title: CD99-Derived Agonist Ligands Inhibit Fibronectin-Induced Activation of β1 Integrin through the Protein Kinase A/SHP2/Extracellular Signal-Regulated Kinase/PTPN12/Focal Adhesion Kinase Signaling Pathway

doi: 10.1128/MCB.00675-16

Figure Lengend Snippet: CD99 peptide suppresses β1 integrin activity via the signaling pathway involving SHP2, RASGAP1, HRAS, RAF1, and ERK1/2. (A, C, D, H) MCF-7 cells were treated with 20 μM CD99CRIII3 for 1 h. Western blot analysis was carried out with the antibodies indicated. β-Actin was used as a loading control. (B) Cells were treated with CD99-Fc I fusion protein or CD99CRIII3 for 1 h, and coimmunoprecipitation of SHP2 was performed with an anti-human CD99 MAb. Immunoprecipitates (IP) and whole-cell lysates (input) were separated by SDS-PAGE and analyzed by Western blotting. (E, F) Cells were dose-dependently treated with the MEK inhibitor PD98059 and incubated with CD99CRIII3 for 1 h. Western blotting was performed with the antibodies indicated. An in vitro cell-matrix adhesion assay was performed to determine the changes in cell adhesion to fibronectin. Statistically significant differences are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01. (G) MCF-7 cells were treated with 3-mer CD99 peptides for 1 h. ERK1/2 phosphorylation was examined by Western blotting.

Article Snippet: An anti-ERK1/2 (3A1) MAb was obtained from Abfrontier (Seoul, South Korea).

Techniques: Activity Assay, Western Blot, SDS Page, Incubation, In Vitro, Cell Adhesion Assay

Journal: Molecular and Cellular Biology

Article Title: CD99-Derived Agonist Ligands Inhibit Fibronectin-Induced Activation of β1 Integrin through the Protein Kinase A/SHP2/Extracellular Signal-Regulated Kinase/PTPN12/Focal Adhesion Kinase Signaling Pathway

doi: 10.1128/MCB.00675-16

Figure Lengend Snippet: CD99-induced PKA activation results in β1 integrin inactivation via regulation of the SHP2/MAPK/ERK signaling pathway. (A, F) In situ PLAs performed to assess the interactions between the pairs of molecules indicated in MCF-7 cells. Numerical values are the average intensities (±SD) of red spots in eight randomly selected fields. (B) Coimmunoprecipitation (Co-IP) assay performed to assess the physical interaction of PKA with SHP2. Immunoprecipitates (IP) and whole-cell lysates (input) were separated by SDS-PAGE and analyzed by Western blotting with the antibodies indicated. (C to E) Western blot analysis was carried out to assess the phosphorylation of ERK1/2 and FAK Y397 and the knockdown efficiency of PKA siRNA. β-Actin was used as a loading control. FN, fibronectin; RCPs, rolling-circle products.

Article Snippet: An anti-ERK1/2 (3A1) MAb was obtained from Abfrontier (Seoul, South Korea).

Techniques: Activation Assay, In Situ, Co-Immunoprecipitation Assay, SDS Page, Western Blot

Journal: Molecular and Cellular Biology

Article Title: CD99-Derived Agonist Ligands Inhibit Fibronectin-Induced Activation of β1 Integrin through the Protein Kinase A/SHP2/Extracellular Signal-Regulated Kinase/PTPN12/Focal Adhesion Kinase Signaling Pathway

doi: 10.1128/MCB.00675-16

Figure Lengend Snippet: PTPN12 and PIN1 act as key mediators in the ERK1/2-mediated regulation of FAK phosphorylation. (A) MCF-7 cells were treated with CD99CRIII3 in the presence or absence of PD98059 for 1 h. In situ PLA was performed to determine the interactions between the protein pairs indicated. Magnification, ×600; scale bars = 10 μm. (B, E) PTPN12 is physically associated with the β1 integrin/FAK complex (B). Endogenous PTPN12 was coimmunoprecipitated (IP) with β1 integrin and FAK but not with SHP2. Lane C, control. Changes in PTPN12 expression and ERK1/2 phosphorylation were confirmed by Western blotting (E). (C) MCF-7 cells were transiently transfected with different amounts of PKA siRNA. Cells were treated with CD99CRIII3 in the presence or absence of KH7 for 1 h. The interaction between PTPN12 and FAK was analyzed by in situ PLA. RCPs, rolling-circle products. (D) Scrambled or PTPN12 siRNA was transiently transfected into MCF-7 cells in a dose-dependent manner. CD99CRIII3-induced modulation of FAK phosphorylation is restored in MCF-7 cells with transient siRNA knockdown of PTPN12. Phosphorylation of FAK at Y397 and S910 was detected with the antibodies indicated, and β-actin was used as a loading control. (F) Cells were transiently transfected with siRNA specifically targeting PTPN12 or PIN1 either alone or combined. Knockdown of endogenous PTPN12 and PIN1 and changes in FAK and ERK1/2 phosphorylation were detected by Western blotting. (G) The interactions between pairs of proteins were analyzed by in situ PLA as indicated. (C, G) Numerical values are the average intensity ± SD of red spots in eight randomly selected fields.

Article Snippet: An anti-ERK1/2 (3A1) MAb was obtained from Abfrontier (Seoul, South Korea).

Techniques: In Situ, Expressing, Western Blot, Transfection