Journal: Journal of Innate Immunity

Article Title: Regulation of TLR10 Expression and Its Role in Chemotaxis of Human Neutrophils

doi: 10.1159/000524461

Figure Lengend Snippet: TLR10 does not affect actin nucleation. a Immunoblots lysates of HL-60-derived neutrophils (2 × 10 6 ). TLR10 gene knockdown was performed as above and cells were stimulated with LPS (1 μg/mL) for 60 min. Actin nucleation proteins ARP3 (1:500) and Diap1 (1:400) were detected in the blot corresponding to the specific molecular weight. Molecular weight is depicted on the left side of the blots. β-Actin showed in the lower panel referred as loading control. b , c Densitometry analysis showed no significant change in actin nucleation proteins.

Article Snippet: Immunoblots were prepared by anti-TLR10 antibody (1:600, 1 mg/mL; Abcam, Cambridge, MA, USA), anti-Diap1 antibody (1:500, Cell Signaling Tech, Danvers, MA, USA), anti-ARP3 antibody (1:400, Cell Signaling Tech, Danvers, MA, USA).

Techniques: Western Blot, Derivative Assay, Knockdown, Molecular Weight, Control

Journal: bioRxiv

Article Title: ERK3/MAPK6 dictates Cdc42/Rac1 activity and ARP2/3-dependent actin polymerization

doi: 10.1101/2022.10.12.511969

Figure Lengend Snippet: (A) Schematic overview of Cdc42-WASP stimulated ARP2/3-dependent actin polymerization based on the cited literature. The process involves: ARP2/3 complex, WASP (VCA) as nucleation promoting factor, filamentous actin (F-actin) and monomeric actin (G-actin). In the initial step Cdc42 is activated by GEF-catalyzed exchange of GDP to GTP. Active Cdc42 (Cdc42-GTP) binds to the GTP-binding domain (GBD) on WASP thereby displacing the VCA domain. While the V-verpolin-like motif binds actin monomer (G-actin), C-central and A-acidic domains bind and activate the ARP2/3 complex. Conformational changes induced by the binding of the ARP2/3 complex promote its binding to the actin filament, which is strengthened by the additional interaction of the ARP2/3 complex with WASP (VCA)-G-actin. Further conformational changes will secure the ARP2/3 complex on the filament and allow its binding to the actin monomer and the polymerization of the newly nucleated filament. Actin polymerizes at the fast-growing/barbed end, elongating towards the plasma membrane and the ARP2/3 complex would cross-link newly polymerizing filament to the existing filament. (B) ERK3 co-precipitates with active Rac1 and Cdc42 in complex with ARP2/3. Active Rac1/Cdc42 pull-down was performed using control and ERK3 knockdown HMECs. Levels of the active Rac1 and Cdc42 were assessed as well as the co-immunoprecipitation levels of ERK3, ARP2, ARP3, and ARPC1A. Levels of the total protein expression were evaluated in the total cell lysates (TCL) and Ponceau S staining was used as a loading control. (C-F) ERK3 regulates F-actin levels in vitro and in vivo . (C) Western Blot analyses of control (CRISPR Co) and ERK3-depleted (CRISPR ERK3) HMECs are presented alongside with representative confocal images of F-actin staining. (D-E) In vivo analysis of F- and G-actin levels in HMECs upon ERK3 knockdown. (D) Representative Western Blot analyses of the enriched F- and G-actin fractions as well as the ERK3 knockdown validation and total actin levels in the total cell lysates (TCL) are presented. (E) F- and G-actin levels were quantified, and ratios were calculated from five (n=5) independent experiments and are presented as mean ± SEM; *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001, unpaired t-test. Analyses of ERK3-dependent regulation of F-actin levels in cancerous MDA-MB231 cells is presented in . Cellular colocalization between endogenous ERK3 and the ARP2/3 was assessed in the absence of Cdc42 and is presented in . (F) Effect of full-length ERK3 on ARP2/3-dependent pyrene actin polymerization was assessed using a pyrene actin polymerization assay. Polymerization induced by the VCA domain of WASP which served as a positive control (green) as well as the ARP2/3 (orange) and ERK3 protein alone (blue) are shown for reference. Actin alone (black) was used to establish a baseline of polymerization. Fluorescence at 360/415 was measured over time and is presented as mean fold change from at least three independent experiments after normalization to the first time point within the respective group. ARP2/3-dependent actin polymerization was measured in the presence of both, ERK3 and WASP (VCA) domain and the results are depicted in .

Article Snippet: Primary antibodies used: phospho-ERK3 (pSer189) (Cat# SAB4504175, Sigma), ARPC1A (Cat# HPA004334, Sigma), ERK3 (Cat #4067, Cell Signaling Technology (CST)), phospho-p44/42 MAPK (Thr202/Tyr204) (Cat# 9101L, CST), V5-tag antibody (Cat # R960-25, Invitrogen), GST antibody (Cat# 2622S, CST), normal rabbit IgG antibody (Cat# 2729, CST), GST (B-14) antibody (Cat# sc-138, Santa Cruz Biotechnology), Rac1 (Cat# 610651, BD Transduction Laboratories), Cdc42 (Cat# 610929, BD Transduction Laboratories), ARP3 (phospho-Ser418) antibody (Cat# orb317559) was obtained from Biorbyt and its specificity was validated in cells overexpressing active (S418D) and phoshpo-impaired (S418A) ARP3 mutants ( ).

Techniques: Binding Assay, Clinical Proteomics, Membrane, Control, Knockdown, Immunoprecipitation, Expressing, Staining, In Vitro, In Vivo, Western Blot, CRISPR, Biomarker Discovery, Polymerization Assay, Positive Control, Fluorescence

Journal: bioRxiv

Article Title: ERK3/MAPK6 dictates Cdc42/Rac1 activity and ARP2/3-dependent actin polymerization

doi: 10.1101/2022.10.12.511969

Figure Lengend Snippet: ERK3 and ARP3 (ARP2/3) colocalization in Cdc42- knockdown cells. HMECs were transfected with two shRNAs targeting Cdc42 (shCdc42#1/#2) as described in the methods section. Afterwards, cells were subjected to either (A) Western Blot analyses to validate to Cdc42 knockdown or (B) IF staining as described in the methods section and confocal imaging to determine localization of ERK3 and ARP3 in the Cdc42 knockdown cells. F-actin was visualized using rhodamine phalloidin to assess cell morphology. (C-D) Graphs present (C) Pearson’s correlation coefficient and (D) Spearman’s rank correlation coefficient values obtained from the co-localization analyses of ERK3 and ARP3 as mean ± SEM from twelve randomly selected cells (n=12).

Article Snippet: Primary antibodies used: phospho-ERK3 (pSer189) (Cat# SAB4504175, Sigma), ARPC1A (Cat# HPA004334, Sigma), ERK3 (Cat #4067, Cell Signaling Technology (CST)), phospho-p44/42 MAPK (Thr202/Tyr204) (Cat# 9101L, CST), V5-tag antibody (Cat # R960-25, Invitrogen), GST antibody (Cat# 2622S, CST), normal rabbit IgG antibody (Cat# 2729, CST), GST (B-14) antibody (Cat# sc-138, Santa Cruz Biotechnology), Rac1 (Cat# 610651, BD Transduction Laboratories), Cdc42 (Cat# 610929, BD Transduction Laboratories), ARP3 (phospho-Ser418) antibody (Cat# orb317559) was obtained from Biorbyt and its specificity was validated in cells overexpressing active (S418D) and phoshpo-impaired (S418A) ARP3 mutants ( ).

Techniques: Knockdown, Transfection, Western Blot, Staining, Imaging

Journal: bioRxiv

Article Title: ERK3/MAPK6 dictates Cdc42/Rac1 activity and ARP2/3-dependent actin polymerization

doi: 10.1101/2022.10.12.511969

Figure Lengend Snippet: (A) Coomassie stained 10% SDS-Page gel with 1 mg of the ARP2/3 protein complex (Cytoskeleton) presenting all the subunits. (B) Binding of increasing concentrations of recombinant GST-ERK3 to the ARP2/3 complex was measured by ELISA as described in the methods section. (C) The interaction between GST-ERK3 and ARP3 was measured in vitro using GST-pull-down assay as described in the methods section. (D) Binding affinity of the recombinant GST-ERK3 protein and ARP3 was assessed by ELISA as described in the methods section and mean absorbance (Abs) ± SEM from three independent experiments is presented. (E) Co-immunoprecipitation (IP) of ARP2/3 protein complex and ERK3 was performed in HMECs using ARP3 antibody. Levels of precipitated ARP3 as well as co-IP of ARP2 and ERK3 were assessed. IgG control was included to determine specificity of the interaction. Total cell lysate (TCL) was included to present expression levels of the verified interacting partners. Ponceau S staining was used as a loading control. (F-G) Actin phenotype of the HMECs was validated upon stable overexpression of the ARP3 non-phosphorylatable (S418A) and the phospho-mimicking (S418D) mutant, respectively. Wild type (WT) ARP3 was used as a control for the mutants and empty vector (EV) served negative control for the overexpression itself. (F) F-actin expression and organization in the negative (S418A) and phospho-mimicking (S418D) ARP3 mutant was visualized by green phalloidin and merged with the Hoechst staining of the nuclei. Four representative confocal images are presented. Images of EV-transfected and ARP3 WT- overexpressing HMECs are presented as controls. (G) Western Blot validation of the overexpression efficiency and phosphorylation of ARP3 at S418. Anti-V5-tag antibody was used to detect levels of exogenous ARP3 WT, S418A and S418D. Expression levels of the endogenous ARP3 were assessed as well as the phosphorylation at S418, total actin was validated. Ponceau S staining was used as a loading control. (H-I) Effect of the ARP3 mutant overexpression on F-actin levels was quantified using F/G actin in vivo assay. (H) Representative Western Blot analyses of F- and G-actin levels detected in fractions obtained from EV, ARP3 WT, S418A, S418D HMECs. (I) Quantification of the F/G actin ratios was performed for three (n=3) independent experiments and is presented as mean ± SEM; *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001, one-way ANOVA, Tukey’s post-test. (J-L) Effect of ERK3 depletion on dense F-actin phenotype of the ARP3 S418D- overexpressing HMECs. HMECs stably overexpressing ARP3 S418D were transduced with lentiviral particles targeting ERK3 (shERK3) and stable knockdown was established as described in the methods section. Cells were further subjected to analyses of the F-actin levels. (J) IF staining with OregonGreen Phalloidin 488 to visualize F-actin levels and organization. Scale bars 28 µm. (K-L) Effect of the ERK3 knockdown on F-actin levels was quantified in the ARP3 S418D mutant overexpressing HMECs using F/G actin in vivo assay. (K) Representative Western Blot analyses of F/G actin levels. ARP3 S418D-(V5-tagged) overexpression and ERK3 knockdown efficiency were validated in TCL. Actin and Ponceau S staining were used as loading controls. (L) Calculated ratios of F/G actin are presented as mean ± SEM from three (n=3) independent experiments; *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001, paired t-test. Colocalization of endogenous ERK3 with endogenous and exogenous ARP3 mutant (S418D) was verified and further effect of the ERK3 depletion on the Rac1 and Cdc42 activity was assessed in ARP3 S418D- overexpressing HMECs and presented in .

Article Snippet: Primary antibodies used: phospho-ERK3 (pSer189) (Cat# SAB4504175, Sigma), ARPC1A (Cat# HPA004334, Sigma), ERK3 (Cat #4067, Cell Signaling Technology (CST)), phospho-p44/42 MAPK (Thr202/Tyr204) (Cat# 9101L, CST), V5-tag antibody (Cat # R960-25, Invitrogen), GST antibody (Cat# 2622S, CST), normal rabbit IgG antibody (Cat# 2729, CST), GST (B-14) antibody (Cat# sc-138, Santa Cruz Biotechnology), Rac1 (Cat# 610651, BD Transduction Laboratories), Cdc42 (Cat# 610929, BD Transduction Laboratories), ARP3 (phospho-Ser418) antibody (Cat# orb317559) was obtained from Biorbyt and its specificity was validated in cells overexpressing active (S418D) and phoshpo-impaired (S418A) ARP3 mutants ( ).

Techniques: Staining, SDS Page, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, In Vitro, Pull Down Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Expressing, Over Expression, Mutagenesis, Plasmid Preparation, Negative Control, Transfection, Western Blot, Biomarker Discovery, Phospho-proteomics, In Vivo, Stable Transfection, Transduction, Knockdown, Activity Assay

Journal: bioRxiv

Article Title: ERK3/MAPK6 dictates Cdc42/Rac1 activity and ARP2/3-dependent actin polymerization

doi: 10.1101/2022.10.12.511969

Figure Lengend Snippet: Detection of the S418 phosphorylation of ARP3 in CRISPR ERK3 HMECs presented in - ).

Article Snippet: Primary antibodies used: phospho-ERK3 (pSer189) (Cat# SAB4504175, Sigma), ARPC1A (Cat# HPA004334, Sigma), ERK3 (Cat #4067, Cell Signaling Technology (CST)), phospho-p44/42 MAPK (Thr202/Tyr204) (Cat# 9101L, CST), V5-tag antibody (Cat # R960-25, Invitrogen), GST antibody (Cat# 2622S, CST), normal rabbit IgG antibody (Cat# 2729, CST), GST (B-14) antibody (Cat# sc-138, Santa Cruz Biotechnology), Rac1 (Cat# 610651, BD Transduction Laboratories), Cdc42 (Cat# 610929, BD Transduction Laboratories), ARP3 (phospho-Ser418) antibody (Cat# orb317559) was obtained from Biorbyt and its specificity was validated in cells overexpressing active (S418D) and phoshpo-impaired (S418A) ARP3 mutants ( ).

Techniques: Phospho-proteomics, CRISPR

Journal: bioRxiv

Article Title: ERK3/MAPK6 dictates Cdc42/Rac1 activity and ARP2/3-dependent actin polymerization

doi: 10.1101/2022.10.12.511969

Figure Lengend Snippet: (A-B) Active Rac1/Cdc42 pull-down according to manufacturer’s protocol (Cat# 16118/19, ThermoFisher) and methods section. (A) Levels of active (GTP-bound) Cdc42 and Rac1 as well as the total protein levels were assessed. Knockdown of ERK3 was validated by ERK3 antibody, exogenous ARP3 (S418D) was detected using a V5-tag antibody and total expression of ARP3 in the cells was determined using an ARP3 antibody. Detection of ARP2 was used as an additional control for the detection of the ARP2/3 complex in both active Rac1/Cdc42 pull-down and TCL. (B) Relative levels of active Cdc42 and Rac1 were calculated with respect to the total protein levels and are presented as mean fold change after normalization with the control (shCo). (C-D) IF staining of ERK3 (secondary antibody: anti-mouse Alexa Fluor 647 (Cat# A21235, ThermoFisher Scientific)) and ARP3 (secondary antibody: Alexa Fluor 488 (Cat# A11008, ThermoFisher Scientific) in (C) WT and (D) ARP3 S418D-overexpressing HMECs. F-actin was visualized using rhodamine phalloidin to assess cell morphology. Scale bar: 21 µm. (E-F) Graphs present (E) Pearson’s correlation coefficient and (F) Spearman’s rank correlation coefficient values obtained from the co-localization analyses of ERK3 and ARP3 as mean ± SEM from twenty-three randomly selected cells (n=23). (G-H) Effect of the ERK3 knockdown on the directional migration of ARP3 S418D- overexpressing HMECs was assessed and quantified using transwell as described in the methods section. Cells were seeded in the inserts in medium without supplements for 1h prior the beginning of the assay. Complete medium was used as a chemoattractant in the lower chamber. (G) Representative images of the analyzed inserts. H) Percentage of the migrated ARP3 S418D shERK3 cells as compared to the control (shCo) is presented as mean ± SEM from three (n=3) independent experiments; *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001, t-test.

Article Snippet: Primary antibodies used: phospho-ERK3 (pSer189) (Cat# SAB4504175, Sigma), ARPC1A (Cat# HPA004334, Sigma), ERK3 (Cat #4067, Cell Signaling Technology (CST)), phospho-p44/42 MAPK (Thr202/Tyr204) (Cat# 9101L, CST), V5-tag antibody (Cat # R960-25, Invitrogen), GST antibody (Cat# 2622S, CST), normal rabbit IgG antibody (Cat# 2729, CST), GST (B-14) antibody (Cat# sc-138, Santa Cruz Biotechnology), Rac1 (Cat# 610651, BD Transduction Laboratories), Cdc42 (Cat# 610929, BD Transduction Laboratories), ARP3 (phospho-Ser418) antibody (Cat# orb317559) was obtained from Biorbyt and its specificity was validated in cells overexpressing active (S418D) and phoshpo-impaired (S418A) ARP3 mutants ( ).

Techniques: Knockdown, Expressing, Control, Staining, Migration