Journal: Frontiers in pharmacology

Article Title: Periplanta americana extract regulates the Th17/Treg cell balance via Notch1 in ulcerative colitis.

doi: 10.3389/fphar.2024.1534772

Figure Lengend Snippet: FIGURE 1 PAE mitigates the symptoms and intestinal barrier damage in UC mice. (A) The schematic design of the experiment. (B) Disease activity index. (C) The colon length. (D) The colon weight/length. (E) Colon pathological scores. (F) Histopathology alterations in the colon (HE, ×200). Red arrows indicate the destruction of colon mucosal. (G) Western blot images of occluding, claudin1, and ZO1 in colon. (H–J) Analysis of gray-scale of occluding, claudin1, and ZO1 protein expression from all groups. (n = 4–8, *P < 0.05 and **P < 0.01 versus control group, #P < 0.05 and ##P < 0.01 versus model group).

Article Snippet: After blocking with 5% skim milk for 1.5 h (h) at room temperature, the membranes were incubated overnight at 4°C with different primary antibodies against Occludin (13409-1- AP, Proteintech), Claudin 1 (13050-1-A, Proteintech), ZO1 (TA5145F, Abmart; 21773-1-A, Proteintech), Foxp3 (Ab215206, Abcam), RORγt (13205-1-AP, Proteintech), Math1 (21215-1-A, Proteintech), Notch1 (T55244S, Abmart; 20687-1-AP, Proteintech), Hes1(11988S, CST) and β-actin (ab179467; Abcam).

Techniques: Activity Assay, Histopathology, Western Blot, Expressing, Control

Journal: Frontiers in pharmacology

Article Title: Periplanta americana extract regulates the Th17/Treg cell balance via Notch1 in ulcerative colitis.

doi: 10.3389/fphar.2024.1534772

Figure Lengend Snippet: FIGURE 5 PAE attenuates the disruption of tight junction proteins in HCoEpic cells by regulating activated Notch1-mediated Th17/Treg imbalance in vitro. (A) Effect of VPA on the viability of Jurkat T cells analyzed with CCK8 assay. (B) Effect of PAE on the viability of Jurkat cells analyzed with CCK-8 assay. (C) Western blot images of Notch1, Math1, RORγt, and Foxp3 in Jurkat T cells. (D–G) Analysis of gray-scale of Notch1, Math1, RORγt, and Foxp3 protein expression. (H) After notch1-activated Jurkat T cells were co-cultured with HCoEpic cells, Western blot images of occluding, claudin1, and ZO1 in HCoEpic cells. (I–K) Analysis of gray-scale of occluding, claudin1, and ZO1 protein expression. (n = 5–8, *P < 0.05 and **P < 0.01 versus control group, #P < 0.05 and ##P < 0.01 versus VPA group).

Article Snippet: After blocking with 5% skim milk for 1.5 h (h) at room temperature, the membranes were incubated overnight at 4°C with different primary antibodies against Occludin (13409-1- AP, Proteintech), Claudin 1 (13050-1-A, Proteintech), ZO1 (TA5145F, Abmart; 21773-1-A, Proteintech), Foxp3 (Ab215206, Abcam), RORγt (13205-1-AP, Proteintech), Math1 (21215-1-A, Proteintech), Notch1 (T55244S, Abmart; 20687-1-AP, Proteintech), Hes1(11988S, CST) and β-actin (ab179467; Abcam).

Techniques: Disruption, In Vitro, CCK-8 Assay, Western Blot, Expressing, Cell Culture, Control

Journal: Journal of Cell Science

Article Title: GIPC3 couples to MYO6 and PDZ domain proteins, and shapes the hair cell apical region

doi: 10.1242/jcs.261100

Figure Lengend Snippet: GIPC3 interaction networks identified through immunoaffinity purification and protein mass spectrometry. (A) Flow chart for anti-GIPC3 immunoaffinity purification from crude chick stereocilia extracts. F/T, flow through. (B–E) Comparison of abundance (riBAQ) of proteins detected in DSP1 total or DTSSP total (starting S7 extract; plotted on X -axis) compared to the immunoprecipitates ( Y -axis) for mouse IgG control (B,C) and 10G5 anti-GIPC3 (D,E) experiments. Panels B and D show results with the DSP-crosslinked starting extract, whereas panels C and E show results with DTSSP crosslinking. Each point represents the average abundance of that protein in two experiments (biological replicates); symbol colors were arbitrarily chosen. Red dashed line is the unity line (equal riBAQ in total and IP). Key proteins are called out. Mouse IgG protein from immunoprecipitation is highlighted in gray. (F) Gene ontology analysis (cellular component) with DAVID of the top 50 proteins from the DTSSP 10G5 eluate. Red, actin-associated components; orange, intermediate filament-associated components; blue, microtubule-associated components; gray, other components. (G) Overlap of protein interaction networks of key proteins from the DTSSP 10G5 eluate (GIPC3, APPL2, MYO6, MYO18A, MYH9 and MYH10). BioGRID-defined protein networks for APPL2, MYO6, MYO18A, MYH9 and MYH10 were compared with the top 100 proteins from the DTSSP 10G5 eluate. Only proteins identified as interactors of two or more of the key proteins were included. Proteins in bold were present in the top 100 proteins from the DTSSP 10G5 eluate.

Article Snippet: Other primary antibodies used were: Atlas Antibody anti-MYO18A (Cat# HPA021121, RRID:AB_1854250; dilution of 1:200 for immunostaining) from Sigma-Aldrich (St Louis, MO, USA); Proteintech (Rosemont, IL, USA) anti-MYO18A (Cat# 14611-1-AP, RRID:AB_2201447; dilution of 1:100); Proteintech anti-APPL2 (Cat# 14294-1-AP, RRID:AB_2878041; dilution of 1:100); Thermo Fisher Scientific (Waltham, MA, USA) anti-TJP1 (also known as ZO1; Cat# 33-9100, RRID:AB_2533147; dilution of 1:200); Santa Cruz Biotechnology (Dallas, TX, USA) anti-LMO7 (Cat# sc-376807, RRID:AB_2892126; dilution of 1:100); Proteintech anti-TARA (TRIOBP; Cat# 16124-1-AP, RRID:AB_2209237; dilution of 1:200); Proteintech anti-ACTN4 (Cat# 19096-1-AP, RRID:AB_10642150; dilution of 1:100); and Biolegend anti-MYH9 (Cat# 909802, RRID:AB_2734686; dilution of 1:200).

Techniques: Immunoaffinity Purification, Mass Spectrometry, Immunoprecipitation

Journal: Journal of Cell Science

Article Title: GIPC3 couples to MYO6 and PDZ domain proteins, and shapes the hair cell apical region

doi: 10.1242/jcs.261100

Figure Lengend Snippet: MYO18A is located in the hair cell apical domain. (A) Top, sequence logo for binding of ligands to GIPC1 PDZ domain; bottom, C-terminal ten amino acids of APPL2, MYO18A, ACTN1 and ACTN4. (B) Immunolocalization of MYO18A in P14.5 mouse cochlea; slices from a three-dimensional image stack. Transects for other image axes are shown in yellow; the X and Y transects in the main X-Y image show the locations for the Y-Z and X-Z images. Arrow indicates concentration of MYO18A immunoreactivity below the IHC cuticular plate. IHC, inner hair cell; IPC, inner pillar cell; OPC, outer pillar cell; OHC, outer hair cell. Immunolocalization experiments for MYO18A were performed more than five times. (C,D) MYO18A immunoreactivity in P15.5 IHCs using lattice SIM imaging. (C) Image showing four IHCs at the stereocilia/cuticular plate level. (D) Image showing a single IHC (labeled with asterisk in C) at the cuticular plate level (different plane than in C). Arrows delineate the gap between cuticular plate actin and the circumferential actin belt. (E,F) MYO18A immunoreactivity in P14.5 IHCs from folded cochleas using Airyscan imaging. E is from a Gipc3 KO /+ mouse and F is from a Gipc3 KO/KO mouse. (G) NanoSPD of MYO18A–GIPC3. Example of filopodial targeting of mCherry–GIPC3 by GFP–FL-MYO18A, mediated by MYO10 NANOTRAP . (H) Expression of GFP–FL-MYO18A and mCherry–FL-GIPC3 constructs in HeLa cells (no MYO10 NANOTRAP expressed). Arrows indicate large cytoplasmic aggregates containing GFP and mCherry. squeezing. Images in C–H representative of at least three repeats. (I) GFP–MYO18A constructs. ‘Motor’, actin- and ATP-binding domains are homologous to myosin motor domains in active myosins; IQ, isoleucine/glutamine calmodulin-binding; PBM, PDZ-binding motif. (J) mCherry–GIPC3 constructs. mCh, mCherry; GH1, GIPC-homology 1; GH2, GIPC-homology 2. (K) Prey (mCh–GIPC3) fluorescence with GFP–MYO18A constructs or GFP control. Mean±s.d. plotted in K and L. One-way ANOVA comparisons to no-bait condition with Dunnett correction for multiple comparisons. Sample sizes ( n ) were GFP–FL-MYO18A (105 filopodia), GFP–IQ-PBM-MYO18A (109), GFP–CC-PBM-MYO18A (116), GFP–C-PBM-MYO18A (129), GFP–ΔPBM-MYO18A (127), GFP (208). (L) Prey (mCh–GIPC3 constructs or mCh–MYO6) fluorescence with GFP-MYO18A constructs or GFP control. One-way ANOVA comparisons to no-bait condition with Dunnett correction for multiple comparisons. Sample sizes ( n ) were mCh–D1-GIPC3 (128 filopodia for GFP–FL-MYO18A and 88 for GFP alone), mCh–D3-GIPC3 (180 and 47) and mCh–MYO6 (107 and 93). Panel widths: B, 37.5 µm for X-Y plot (same scale applies to Y-Z and X-Z panels); C, 50 µm; D, 12 µm; E-F, 45 µm; G,H, 15 µm.

Article Snippet: Other primary antibodies used were: Atlas Antibody anti-MYO18A (Cat# HPA021121, RRID:AB_1854250; dilution of 1:200 for immunostaining) from Sigma-Aldrich (St Louis, MO, USA); Proteintech (Rosemont, IL, USA) anti-MYO18A (Cat# 14611-1-AP, RRID:AB_2201447; dilution of 1:100); Proteintech anti-APPL2 (Cat# 14294-1-AP, RRID:AB_2878041; dilution of 1:100); Thermo Fisher Scientific (Waltham, MA, USA) anti-TJP1 (also known as ZO1; Cat# 33-9100, RRID:AB_2533147; dilution of 1:200); Santa Cruz Biotechnology (Dallas, TX, USA) anti-LMO7 (Cat# sc-376807, RRID:AB_2892126; dilution of 1:100); Proteintech anti-TARA (TRIOBP; Cat# 16124-1-AP, RRID:AB_2209237; dilution of 1:200); Proteintech anti-ACTN4 (Cat# 19096-1-AP, RRID:AB_10642150; dilution of 1:100); and Biolegend anti-MYH9 (Cat# 909802, RRID:AB_2734686; dilution of 1:200).

Techniques: Sequencing, Binding Assay, Concentration Assay, Imaging, Labeling, Expressing, Construct, Fluorescence

Journal: International Journal of General Medicine

Article Title: The Predictive Value of Serum DAO, HDC, and MMP8 for the Gastrointestinal Injury in the Early Stage of Acute Pancreatitis in an Animal Model and a Clinical Study

doi: 10.2147/IJGM.S461352

Figure Lengend Snippet: Serum DAO, HDC, and MMP8 levels and tight junction protein expression in AP rats. ( A–C ) Serum DAO, HDC, and MMP8 levels were higher in the AP group than in the normal group and SO group at the same point of time. ( D ) Western blot for tricellulin, claudin-1, ZO-1, and occludin in the intestinal mucosa of rats. ( E–H ) The expression of tricellulin, claudin-1, ZO-1, and occludin was significantly decreased in the AP group than in the normal group and SO group.

Article Snippet: The membrane was blocked with 5% skimmed milk at 22°C for 1 h. The membrane with proteins was incubated with primary antibodies against GAPDH (1:5000 dilution, Abcam, USA), tricellulin (1:1000 dilution, ABclonal, Wuhan, China), claudin-1 (1:800 dilution, Proteintech, Wuhan, China), ZO-1 (1:1000 dilution, Proteintech, Wuhan, China), and occludin (1:800 dilution, Proteintech, Wuhan, China) overnight at 4°C.

Techniques: Expressing, Western Blot