Journal: Respiratory Research

Article Title: FGF18 alleviates sepsis-induced acute lung injury by inhibiting the NF-κB pathway

doi: 10.1186/s12931-024-02733-1

Figure Lengend Snippet: FGF18 promotes HUVECs repair by inhibiting NF-κB p65 activation. (A) HUVECs transfected with si- FGF18 were then treated with JSH-23 (20 µM) for 3 h, HUVECs were subjected to western blotting analysis. The expression of TNF-α and IL-6 were detected. ( n = 5 per group). (B) HUVECs transfected with si- FGF18 were then treated with Bay11-7082 (10 µM) for 3 h, HUVECs were subjected to western blotting analysis. The expression of TNF-α and IL-6 were detected. ( n = 5 per group). (C) Immunofluorescent staining of ICAM-1/VCAM-1 (red) and DAPI (blue) in HUVECs were detected. ( n = 3 per group, Scale bar = 50 μm). (D) Immunofluorescent staining of ICAM-1/VCAM-1 (red) and DAPI (blue) in HUVECs were detected. ( n = 3 per group, Scale bar = 50 μm)

Article Snippet: U0126 (CAS: HY-12,031) and Bay11-7082 (CAS: 19542-67-7) was purchased from Med Chem Express (New Jersey USA).

Techniques: Activation Assay, Transfection, Western Blot, Expressing, Staining

Journal: ACS synthetic biology

Article Title: Analysis of Three Architectures for Controlling PTP1B with Light.

doi: 10.1021/acssynbio.1c00398

Figure Lengend Snippet: Figure 2. Analysis of natively localized constructs. (A) Images of COS-7 cells expressing RFP-tagged variants of PTP1B: PTP1B435 is the wild-type protein, which contains a C-terminal region (a disordered proline-rich domain followed by a short membrane anchor) that localizes it to the endoplasmic reticulum (ER). PTP1BPS* is a version of PTP1BPS that includes residues 299−435 from the wild-type enzyme attached to its C- terminus; I1* and S1A* include the same region of the full-length enzyme. PTP1B435, PTP1BPS*, and I1* exhibit indistinguishable localization patterns that are consistent with localization to the ER; for S1A*, fluorescence in the cytosol suggests that a significant fraction of its N-terminal half (i.e., RFP-PTP1B-Zdk1) is dissociated from its C-terminal half (i.e., ER-localized LOV2-PTP1B; scale bar, 10 μm). Additional images appear in Figure S3. (B,C) ELISA-based measurements of IR phosphorylation in HEK293T/17 stably expressing variants of PTP1B after 10 min exposure to light (455 nm) or darkness. The DR (i.e., the ratio of light- to dark-state signals) appears above the bars for each construct. (B) Illumination increases IR phosphorylation for all constructs. (C) The introduction of LOV2-stabilizing mutations does not improve the dynamic range of S1A*. The plotted data depict the mean and SE for measurements of n = 3 biological replicates. The error in DR depicts propagated SE for n = 3 biological replicates under each condition (light and dark). Source data appear in Table S6.

Article Snippet: We exposed cells to (i) 455 nm light, (ii) total darkness, or (iii) 300 μM of BBR for 10 min and immediately removed the media and lysed the cell in lysis buffer (Cell Signaling Technology) supplemented with 1X halt phosphatase inhibitor and 1X halt protease inhibitor (Thermo Fisher Scientific) for 10 min. We spun the cells down, collected the supernatant, and diluted the samples to 30 mg/mL for the PathScan PhosphoInsulin Receptor β (panTyr) Sandwich ELISA Kit (Cell Signaling Technology; #7082).

Techniques: Construct, Expressing, Membrane, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Stable Transfection

Journal: ACS synthetic biology

Article Title: Analysis of Three Architectures for Controlling PTP1B with Light.

doi: 10.1021/acssynbio.1c00398

Figure Lengend Snippet: Figure 3. Extension of the insertion-based approach to other PTPs. (A) Aligned crystal structures of the catalytic domains of PTP1B, SHP2, and TCPTP (pdb entries 2cm2, 3zm1, and 1l8k, respectively). Highlights: competitive inhibitor (red spheres, pdb entry 2f71), insertion site I1 (PTP1B205−206, TCPTP206−207, and SHP2449−450; red sticks), and insertion site I2 (PTP1B186−187, red sticks). (B) Initial rates of SHP2I1-catalyzed hydrolysis of pNPP in the presence and absence of light (455 nm). Values of kcat and KM are indistinguishable (p < 0.05). (C) ELISA-based measurements of IR phosphorylation in HEK293T/17 cells stably expressing photoswitchable variants of TCPTP after 10 min exposure to light (455 nm) or darkness. TCPTPPS* is a version of TCPTPPS that includes residues 318−415 from the wild-type enzyme (i.e., the ER anchor) attached to its C- terminus; TCPTPI1* and TCPTPI2* include the same region of the full-length enzyme. Illumination increases IR phosphorylation for TCPTPPS* and TCPTPI1*, but not TCPTPI2*. The plotted data depict the mean and SE for measurements of n = 3 biological replicates. The DR appears above the bars for each construct; here, error depicts propagated SE for n = 3 biological replicates under each condition (light and dark). Tables S1 and S6 provide source data.

Article Snippet: We exposed cells to (i) 455 nm light, (ii) total darkness, or (iii) 300 μM of BBR for 10 min and immediately removed the media and lysed the cell in lysis buffer (Cell Signaling Technology) supplemented with 1X halt phosphatase inhibitor and 1X halt protease inhibitor (Thermo Fisher Scientific) for 10 min. We spun the cells down, collected the supernatant, and diluted the samples to 30 mg/mL for the PathScan PhosphoInsulin Receptor β (panTyr) Sandwich ELISA Kit (Cell Signaling Technology; #7082).

Techniques: Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Stable Transfection, Expressing, Construct