Journal: Oxidative Medicine and Cellular Longevity

Article Title: Apolipoprotein (a)/Lipoprotein(a)-Induced Oxidative-Inflammatory α 7-nAChR/p38 MAPK/IL-6/RhoA-GTP Signaling Axis and M1 Macrophage Polarization Modulate Inflammation-Associated Development of Coronary Artery Spasm

doi: 10.1155/2022/9964689

Figure Lengend Snippet: Lp(a) promotes inflammation in PMDMs and HCASMCs by inducing α 7-nAChR-dependent activation of p38 MAPK signaling. (a) Representative western blot photo images showing the effect of treating patient monocyte-derived macrophages with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on α 7-nAChR, IL-6, p-p38 MAPK, and p38 MAPK protein expression levels. (b) Representative western blot photo images showing the effect of treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min ( upper panel ) or with 1 μ M Lp(a) at 15, 30, and 60 min time points ( lower panel ), on RhoA-GTP, RhoA, p-p38 MAPK, and p38 MAPK protein expression levels. (c) Representative western blot photo images showing that treating HCASMCs with 0.5 μ M–2 μ M Lp(a) for 60 min increases ROCK activity dose dependently. (d) Representative western blot images showing how shCHRNA7 affects the expression of RhoA-GTP, RhoA, α 7-nAChR, p-p38 MAPK, and p38 MAPK in HCASMCs in the presence or absence of 1 μ M Lp(a). Histograms show the effect of shCHRNA7 on CD80 MFI in HCASMCs in the presence or absence of 1 μ M Lp(a). (e) PMDMs were treated with different concentrations of Lp(a) (0-2 μ M) and the nitric oxide production was measured. (f) Lp(a) treatment dose dependently reduced the iNOS expression level in PMDMs. HCASMC: human coronary artery smooth muscle cell; MFI: median fluorescence intensity; PMDM: patient monocyte-derived macrophage; RhoA: Ras-homologous A; ROCK: Rho-kinase; shCHRNA7: α 7-nAChR-targeting short hairpin RNA. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; GAPDH served as loading control.

Article Snippet: Western blot analyses were performed according to standard protocol [ ] using the following antibodies against: α 7-nAChR (ab216485; 1 : 1000), p38 (ab31828; 1 : 1000), p-p38 (phospho T180+Y182) (ab4822; 1 : 1000), IL-6 (ab9324; 1 : 1000), inducible NO synthase (ab3523; 1 : 1000), and GAPDH (ab9484; 1 : 10,000), purchased from Abcam (Abcam plc., Cambridge, UK), and RhoA (#2117; 1 : 1000), RhoA-GTP (#8820; 1 : 1000), ROCK1 (#4035; 1 : 1000), ROCK2 (#9029; 1 : 1000), t-MBS (#2634; 1 : 1000), and p-MBS (#3040; 1 : 1000) from Cell Signaling Technology (Cell Signaling, Danvers, MA, USA) in Supplementary Table .

Techniques: Activation Assay, Western Blot, Derivative Assay, Expressing, Activity Assay, Fluorescence, shRNA

Journal: Nature Communications

Article Title: The lysosomal Ragulator complex plays an essential role in leukocyte trafficking by activating myosin II

doi: 10.1038/s41467-021-23654-3

Figure Lengend Snippet: a MLC phosphorylation in the presence or absence of Lamtor1. WT-THP1, Lamtor1 -KO-THP1, and Lamtor1 -KO-Full-THP1 cells were lysed, and MLC phosphorylation was detected by western blotting with anti-p-MLC antibody (left). The concentration of p-MLC of WT-THP1 (white bar), Lamtor1 -KO-THP1 (black bar), and Lamtor1 -KO-Full-THP1 (mesh bar) in SDS-PAGE gel bands was determined using ImageJ, and statistical analysis was performed (right). b , c Myosin II-dependent Lamtor1-mediated motility. Chemotaxis of WT-THP1 (white bar), Lamtor1 -KO-THP1 (black bar), Lamtor1 -KO-Full-THP1 (mesh bar), blebbistatin-treated WT-THP1 (diagonal bar), and blebbistatin-treated Lamtor1 -KO-Full-THP1 (dark mesh bar) in response to 25 ng/ml CCL2 was determined by Transwell assay (pore size, 5 μm) ( b ). Chemotaxis of WT-THP1 (white bar), Lamtor1 -KO-THP1 (black bar), and Lamtor1 -KO-THP1 expressing MLC-DD-HA, a constitutively active form of MLC (mesh bar) in response to CCL2 were evaluated by a Transwell assay (pore size, 5 μm) (left). Expression of MLC-DD-HA in Lamtor1 -KO-THP1 was evaluated by western blotting using an anti-HA antibody (right) ( c ). d Active form of RhoA in WT and Lamtor1 −/− BMDCs. Cells were lysed, and GTP-bound RhoA was pulled down with GST-Rhotekin-RBD and detected by western blotting with an anti-mouse RhoA antibody. e MYPT1 phosphorylation in WT and Lamtor1 −/− DCs. WT and Lamtor1 −/− BMDCs were lysed and phosphorylated MYPT1 was evaluated by western blotting with an anti-p-MYPT1 antibody. Statistical analysis was performed by two-sided ANOVA with Tukey’s post hoc test ( a – c ) [means ± s.d.; *p < 0.05, **p < 0.01, ***p < 0.001]. Data are representative of three experiments.

Article Snippet: After BMDC lysis, the active form of RhoA was pulled down with GST-Rhotekin-RBD fusion protein and immunoprecipitated with glutathione resin (Active RhoA Detection Kit, Cell Signaling Technologies).

Techniques: Western Blot, Concentration Assay, SDS Page, Chemotaxis Assay, Transwell Assay, Expressing

Journal: Cancers

Article Title: CD99–PTPN12 Axis Suppresses Actin Cytoskeleton-Mediated Dimerization of Epidermal Growth Factor Receptor

doi: 10.3390/cancers12102895

Figure Lengend Snippet: FAK functions as a critical mediator in EGF-induced activation of Rac1 and RhoA GTPases during EGFR signaling. ( A , C ) MCF-7 cells stimulated by binding of ligand to its receptor were analyzed for activation of small GTPases. Activated GTP-bound Rac1 or RhoA in the cell lysates were determined by immunoblotting with anti-Rac1 or anti-RhoA antibodies. β-actin was used as a loading control. ( B , D ) MDA-MB-231 cells were transfected with CA-FAK or FAK Y397F plasmids and incubated in the presence or absence of 25 ng/mL EGF at 37 °C, 5% CO 2 for 15 min. The interactions between the pairs of molecules indicated were assessed by in situ PLA. *** p < 0.001. ( E ) Activation of small GTPases in MCF-7 cells was determined by immunoblotting. ( F ) EGFR dimerization in MDA-MB-231 cells was assessed by in situ PLA and the experiments were duplicated. ( G ) EGFR endocytosis in MCF-7 cells was determined by IFA as described above. Original magnification of representative images, 600×. Scale bars = 10 μm.

Article Snippet: Active GTPase assay was performed using an active Rac1 or RhoA detection kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions.

Techniques: Activation Assay, Binding Assay, Western Blot, Transfection, Incubation, In Situ

Journal: Cancers

Article Title: CD99–PTPN12 Axis Suppresses Actin Cytoskeleton-Mediated Dimerization of Epidermal Growth Factor Receptor

doi: 10.3390/cancers12102895

Figure Lengend Snippet: Modulation of actin polymerization by Rac1/RhoA GTPases is essential for EGF-induced dimerization and endocytosis of EGFR. ( A , E ) The changes in the activation of Rac1/RhoA-mediated signaling were observed in MCF-7 cells. The interactions between the pairs of molecules indicated were assessed by in situ PLA. *** p < 0.001. ( B , F ) To determine EGFR dimerization, MDA-MB-231 cells were subjected to BS 3 chemical-mediated crosslinking, as described above and in the Materials and Methods. Cell extracts were assessed via Western blotting to determine the dimerization and phosphorylation levels of EGFR and the expression levels of indicated proteins. β-actin was used as a loading control. EGFR endocytosis ( C , G ) and actin cytoskeleton organization ( D , H ) in MCF-7 cells transfected with siRNAs specific for ARP2 and Ezrin or plasmids encoding CA-GTPases or CA-FAK. Original magnification of representative images, 600×. Scale bars = 10 μm.

Article Snippet: Active GTPase assay was performed using an active Rac1 or RhoA detection kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions.

Techniques: Activation Assay, In Situ, Western Blot, Expressing, Transfection