Journal: Cell Communication and Signaling : CCS

Article Title: ICAM-2 regulates vascular permeability and N-cadherin localization through ezrin-radixin-moesin (ERM) proteins and Rac-1 signalling

doi: 10.1186/1478-811X-12-12

Figure Lengend Snippet: ERMs and Rac1 activity are involved in ICAM-2-dependent NCad recruitment and cell-cell contact. A - Rac1 activity in MCEC cell lines, as measured by the GST-PAK pull-down assay. (a) Representative Western blot analysis of Rac1 activity. Rac1-GTP pull-down with GST-PAK as well as total Rac1 were detected using mAb Cl23A8. (b) Quantification of Rac1 activity (Rac1-GTP/Total Rac1) in IC2 neg, IC2 FL, IC2 ΔERM, IC2 ΔTAIL cell lines. Error bars indicate mean ± s.e.m., n = 6. Statistical analysis (t-test: *p < 0.05, **p < 0.005). B - Effect of DN and CA Rac1 on the morphology and the distribution of NCad in the IC2 neg and IC2 FL lines. IC2 neg or IC2 FL were transfected with pEGFP control ( a-d and m-p ), pEGFP-V12 Rac1 ( e-h and q-t ) or pEGFP-V12-N17 Rac1 ( i-l and u-x ). Cells were co-stained for ICAM-2 and NCad. ICAM-2 was stained using the mAb 3C4 anti-mouse ICAM-2 followed by anti-rat AlexaFluor488 (pseudo colored Red). NCad was stained using mAb Cl32 anti-NCad followed by anti-mouse AlexaFluor555 (pseudo colored Blue). Bar = 25 μm.

Article Snippet: Dejana (IFOM, Italy [ ]). mAb PECAM-1 P2B1 was purchased from Santa-Cruz. mAb MJ7/18 anti-mouse endoglin was purchased from Chemicon. mAb Cl 23A8 anti-Rac1 was purchased from Transduction Laboratories.

Techniques: Activity Assay, Pull Down Assay, Western Blot, Transfection, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: ICAM-2 regulates vascular permeability and N-cadherin localization through ezrin-radixin-moesin (ERM) proteins and Rac-1 signalling

doi: 10.1186/1478-811X-12-12

Figure Lengend Snippet: In vitro and in vivo cell permeability assays. A - Cells were grown in full medium on gold electrodes until stable impedance was reached. TEER changes were recorded after 48 h and changes in TEER at baseline or B - after 10 min thrombin stimulation. Shown are average values -/+ s.e.m. of 4 independent experiments. C - Cells were grown in full medium on gold electrodes until stable impedance was reached. Cells were then infected with recombinant adenovirus encoding constitutively active (DA) or dominant negative (DN) myc-Rac1 at an m.o.i. of 400. After 4 h the virus was removed and cells starved to assess barrier development. TEER changes were recorded after 48 h. Shown are average values -/+ s.e.m of 4 independent experiments. D - In vivo permeability assay: albumin leakage in postcapillary venules from the cremaster muscles of WT and ICAM-2 KO (IC2 neg) mice in response to thrombin (2U/mL) during 10 min. Data are mean ± s.e.m. of n = 6 mice per group. t-test WT vs IC2-/- * p < 0.05, **p < 0.01.

Article Snippet: Dejana (IFOM, Italy [ ]). mAb PECAM-1 P2B1 was purchased from Santa-Cruz. mAb MJ7/18 anti-mouse endoglin was purchased from Chemicon. mAb Cl 23A8 anti-Rac1 was purchased from Transduction Laboratories.

Techniques: In Vitro, In Vivo, Permeability, Infection, Recombinant, Dominant Negative Mutation

Journal: Frontiers in Pharmacology

Article Title: Osthole Ameliorates Estrogen Deficiency-Induced Cognitive Impairment in Female Mice

doi: 10.3389/fphar.2021.641909

Figure Lengend Snippet: List of antibodies used in the study.

Article Snippet: Rac 1 , 23A8 , EMD-millipore corp.

Techniques: Transduction

Journal: Cancer cell

Article Title: RAC1 P29S Induces a Mesenchymal Phenotypic Switch via Serum Response Factor to Promote Melanoma Development and Therapy Resistance.

doi: 10.1016/j.ccell.2019.05.015

Figure Lengend Snippet: Figure 1. Effect of Activation of RAC1P29S on Survival of Melanocytes (A) Schematic of the ER-RAC1P29S fusion protein system. ER, estrogen receptor; 4OHT, 4-hydroxytamoxifen; HSP, heat shock protein. (B) RAC1-GTP assay in MCF10A cells (left) and mouse melanocyte cell line melan-a (right) stably expressing ER-RAC1P29S. Cells were treated with 500 nM 4OHT for 24 h and ER-RAC1P29S binding to the PAK1 RAC1 binding domain was assessed using a pull-down assay. (C) PAK1/2 and ERK1/2 phosphorylation by ER-RAC1P29S in melanocytes. Cells were treated with 500 nM 4OHT for 24 h. (D) Quantification of phospho-PAK1/2 and phospho-ERK1/2 levels detected by immunoblotting, normalized using vinculin loading control (n = 3 or more in- dependent experiments); t test versus 0 h was used with Holm-Sidak correction for multiple testing; *p < 0.05, ***p < 0.001; n.s., not statistically significant. (E) Morphological changes induced by ER-RAC1P29S activation in melan-a melanocytes treated with 500 nM 4OHT. (F) Viability of melan-a melanocytes with ER-RAC1P29S in growth factor-reduced medium (fetal calf serum [FCS] 0.25%, 12-O-tetradecanoylphorbol-13-acetate [TPA]-free). Cells were treated with 1 mM 4OHT and viability was quantified using CellTiter-Glo (n = 3 independent experiments); t test was used for statistical comparison. (G) Soft agar sphere formation of melan-a melanocytes with activated ER-RAC1P29S. Cells were grown in full medium containing 1 mM 4OHT for 3 weeks before staining, imaging, and automated counting (n = 3 biological replicates). (H) Effect of activation of ER-RAC1P29S on apoptosis in melan-a melanocytes. Cells were treated with 1 mM 4OHT. Cleaved caspase-3 was quantified and normalized to cell viability (n = 2 independent experiments, three biological replicates per experiment). Bars represent means ± SD. See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies phospho-AKT (T308) CST #13038 phospho-AKT (S473) CST #9271 phospho-AKT (S473) CST #4060 AKT CST #2920 phospho-ERK1/2 (T202/T204) CST #9101 ERK1/2 CST #9107 phospho-PAK1/2 (S199/192) CST #2605 phospho-PAK1/2 (T423/402) CST #2601 PAK1/2/3 CST #2604 phospho-MEK1/2 (S217/221) CST #9145 RAC1 (23A8) Merck Millipore 05-389 Vinculin (hVIN-1) Sigma-Aldrich V9131 Myc (9E10) In-house (CRUK LRI) N/A Vimentin (EPR3776) Abcam ab92547 N-cadherin CST #13116 c-Jun CST #9165 phospho-c-Jun (S73) CST #9164 ER (MC-20) Santa Cruz sc-542 Fibronectin Abcam ab2413 phospho-BRAF (S445) CST #2696 phospho-CRAF (S338) CST #9427 S100 (4C4.9) Abcam ab4066 SOX10 (A2) Santa Cruz sc-365692 p16 (EPR1473) Abcam ab108349 p27 (F-8) Santa Cruz sc-1641 B220 (RA3-6B2) BD Biosciences 553086 B220-BV510 Biolegend RA3-6B2 CD19-BV605 Biolegend 6D5 CD38-APC Biolegend 90 CD95-BV421 BD Biosciences Jo2 CD138-BV786 BD Biosciences 281-2 NK1.1-PE Biolegend PK136 CD44-APC Ebioscience IM7 CD62L-PeCy7 Ebioscience MEL-14 CD3e-PerCPEFluor710 Ebioscience 145-2C11 CD4-BV421 Biolegend GK1.5 CD8a-FITC Ebioscience 53-6.7 PTEN CST #9559 Chemicals, Peptides, and Recombinant Proteins 4OH-tamoxifen Sigma-Aldrich H6278 Tamoxifen Sigma-Aldrich T5648 CCG-1423 Selleckchem S7719 CCG-203971 Tocris 5277 (Continued on next page) e1 Cancer Cell 36, 68–83.e1–e9, July 8, 2019

Techniques: Activation Assay, Stable Transfection, Expressing, Binding Assay, Pull Down Assay, Phospho-proteomics, Western Blot, Control, Comparison, Staining, Imaging

Journal: eLife

Article Title: Shigella entry unveils a calcium/calpain-dependent mechanism for inhibiting sumoylation

doi: 10.7554/elife.27444

Figure Lengend Snippet: Figure 2. Shigella inhibits sumoylation by a calpain-dependent mechanism. (A) HeLa cells were pretreated by vehicle (DMSO) or 100 mM MDL28170 for 1 hr and then left uninfected or infected with the M90T strain or the mxiD mutant for 2 hr. Immunoblot analysis were performed using anti-Capns1 (Calpain small subunit 1), - calpastatin, -SUMO1, -SUMO2/3 and -tubulin antibodies. The 18 kDa truncated Capns1 fragment is indicated by Figure 2 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.27444 17 of 24 Continued Reagent type (species) or resource Designation Source or reference Identifiers cell line (HT1080) UBC9-auxin degron this paper N/A genetic reagent (siRNA) control Dharmacon #D-001810–10 genetic reagent (siRNA) Capns1 Dharmacon #L-009979–00 genetic reagent (siRNA) Ubc9 Dharmacon #L-004910–00 genetic reagent (siRNA) SAE2 Dharmacon #L-005248–01 genetic reagent (siRNA) RhoGDIa Dharmacon #L-016253–00 transfected construct (Plasmid) pEGFP-RhoGDIa WT PMID: 22393046 N/A transfected construct (Plasmid) pEGFP-RhoGDIa K138R PMID: 22393046 N/A antibody anti-SAE1 Abcam #ab97523/AB_10681015 antibody anti-SAE2 Abcam #ab22104/AB_446785 antibody anti-SUMO1 Abcam Y299/AB_778173 antibody anti-SUMO2/3 Abcam 8A2/AB_1658424 antibody anti-UBC9 Abcam EP2938Y/AB_1267373 antibody anti-SUMO1 DSHB Iowa 21C7/AB_2198257 antibody anti-Calpastatin Cell Signaling Technology #4146/AB_2244162 antibody anti-SAE2 Cell Signaling Technology D15C11/AB_10889561 antibody anti-Cdc42 Cell Signaling Technology 11A11/AB_10695738 antibody anti-RhoA Cell Signaling Technology 67B9/AB_10693922 antibody anti-Tubulin Cell Signaling Technology DM1A/AB_1904178 antibody anti-RanGAP1 Santa Cruz C-5/AB_2176987 antibody anti-GFP Santa Cruz C-2 antibody anti-RhoGDIa Merck Millipore #06–730/AB_310229 antibody anti-Capns1 Merck Millipore MAB3083/AB_2070014 antibody anti-Rac1 Merck Millipore 23A8/AB_309712 antibody anti-LPS PMID: 25097252 N/A antibody anti-SP100 PMID: 7559785 N/A chemical compound, drug Phalloidin–Tetramethylrhodamine B isothiocyanate Sigma P1951 / AB_2315148 chemical compound, drug Dapi Sigma D9542 chemical compound, drug Cytochalasin D Sigma C8273 chemical compound, drug MDL 28170 Sigma M6690 chemical compound, drug Ionomycin Sigma I3909 chemical compound, drug BAPTA-AM Enzo life sciences BML-CA411-0025 chemical compound, drug Indole-3-acetic acid Sigma I5148 chemical compound, drug N-Ethylmaleimide Sigma E3876 peptide, recombinant protein Recombinant SAE2 Novus biologicals NBP2-50574-20ug peptide, recombinant protein Recombinant Calpain-1 Merck Millipore 208712 peptide, recombinant protein GST-SENP2cat this paper NP_06760.2 peptide, recombinant protein SUMO1-AMC Boston Biochem UL-551 peptide, recombinant protein SUMO2-AMC Boston Biochem UL-758 software, algorithm Icy software Institut Pasteur PMID: 22743774 Bacterial strains and cell culture Shigella flexneri serotype 5a strains were isolated on congo red agar plates.

Techniques: Infection, Mutagenesis, Western Blot