anti p mlc2  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Plastin-3 membrane recruitment drives cell-in-cell invasion during entosis

doi: 10.64898/2026.03.17.709257

Figure Lengend Snippet: (A) Schematic overview of ROCK1 construct transfection used to assess entosis induction. (B) Representative immunoblots showing ROCK1, phosphorylated MLC2 (pMLC2), and tubulin (loading control) in MCF7 cells transfected with GFP, GFP–ROCK1, or constitutively active GFP–ROCK1 Δ3. Immunoblot analysis of pMLC2 was performed 6 h post-transfection, whereas ROCK1 expression was assessed at 24 h. (C) Representative 3D confocal images of live MCF7 cells expressing GFP–ROCK1 Δ3 and stained with SiR-Actin (red) and Hoechst (blue). White arrows indicate entotic structures. Boxed regions showing representative entotic structures are magnified, with corresponding orthogonal z-stack views shown alongside each image. (D) Quantification of entotic events in MCF7 cells expressing GFP–ROCK1 or GFP–ROCK1 Δ3. (E) Distribution percentages of GFP-positive cells participating in CIC structures as outer cells, inner cells, or both. (F) Representative time-lapse imaging (phase contrast, Hoechst, and GFP) capturing an entotic event in MCF7 cells expressing GFP–ROCK1 Δ3. Yellow dotted lines outline inner cells, and red dotted lines outline host cells. Experiments were performed in biological triplicate (n = 3). Data are presented as mean ± SEM. Statistical analysis in panel D was performed using a paired two-tailed Student’s t-test (**p < 0.01), whereas panel E was analyzed using two-way ANOVA followed by Bonferroni post hoc test (*p < 0.05, **p < 0.01, ****p < 0.0001).

Article Snippet: Membranes were blocked in 5% non-fat milk in TBS-T or in 5% BSA in TBS-T when detecting phospho-specific antibodies and incubated with primary antibodies against ROCK1 (rabbit monoclonal, 1:1000; Cell Signaling Technology, #28999S), RhoA (rabbit monoclonal, 1:1000; Cell Signaling Technology, #2117S), PLS2 (rabbit monoclonal, 1:1000; Atlas Antibodies, #HPA019493), PLS3 (mouse monoclonal, 1:1000; Invitrogen, #MA5-27772), α-tubulin (rabbit polyclonal, 1:3000; Cell Signaling Technology, #2144S), phospho-MLC2 (Ser19) (rabbit, 1:1000; Cell Signaling Technology, #3671S), and GFP (chicken polyclonal, 1:1000; Rockland Immunochemicals, #600-901-215).

Techniques: Construct, Transfection, Western Blot, Control, Expressing, Staining, Imaging, Two Tailed Test

Journal: Foods

Article Title: Bacteroides finegoldii and Parabacteroides goldsteinii Mediate Fucoidan-Induced Attenuation of Intestinal Inflammation in Mice Through Betaine- and Spermidine-Related Pathways

doi: 10.3390/foods15020203

Figure Lengend Snippet: PG and BF suppressed activation of the MLCK/MAPK pathways in colon tissues. ( A ) Representative Western blot images and quantitation of MLCK expression and p-MLC2/MLC2 ratio. ( B ) Representative Western blot analysis and quantitation of MAPK pathway-related proteins. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Membranes were blocked for 1 h, washed, and incubated overnight at 4 °C with diluted primary antibodies: anti-TLR4 (38519), p65 (8242T), p-IκBα (2859T), p-p38 (8690), p-p65 (3033T), p38 (4511), p-MLC2 (3671T), ZO-1 (8193), and claudin-1 (4933) (CST, Danvers, MA, USA), and anti-occludin (ab216327), MLCK (ab232949), MLC2 (ab92721), IκBα (ab76429), JNKs (ab179462), p-JNKs (ab124956), ERKs (ab184699), and p-ERKs (ab201015) (Abcam, Cambridge, UK).

Techniques: Activation Assay, Western Blot, Quantitation Assay, Expressing

Journal: The FASEB Journal

Article Title: TRPV4 ‐Mediated Mechanosensing Regulates the Endothelial‐to‐Mesenchymal Transition: Implications for Atherosclerosis

doi: 10.1096/fj.202403198R

Figure Lengend Snippet: Phospho‐MLC2 is involved in TRPV4‐mediated EndMT and N‐terminal 100–130 amino acid residues of TRPV4 are required for phosphorylation of MLC2. (A) TRPV4 KO aECs were culture on stiff (50 kPa) PA hydrogels. Representative images from five different fields per condition showing morphological changes and co‐localization of F‐Actin (green) and p‐MLC2 (red) with DAPI (blue) staining in TGFβ1 (5 ng/mL)‐stimulated TRPV4 KO aECs transfected with Ad‐TRPV4‐WT (with or without GSK219), Ad‐TRPV4‐Δ1‐30, Ad‐TRPV4‐Δ1‐130, Ad‐TRPV4‐Δ100‐130, or Ad‐Vec. (B, C) Quantitation of results from Figure A. Data are expressed as mean ± SEM, n = 20 cells/condition; 1‐way ANOVA followed by Bonferroni test; *** p < 0.001, **** p < 0.0001. (D) WT and TRPV4 KO aEC were treated with TGFβ1 (5 ng/mL) for 30 and 60 min or kept untreated (UT) for the control group. Immunoblotting assay and quantification of p‐MLC2 (Thr18/Ser19) relative to total MLC2 protein levels are shown. All experiments performed three times. (E) Bar graphs show quantification of results from D. Data analyzed using One‐way ANOVA followed by Bonferroni's multiple comparison test; *** p < 0.001. (F) TRPV4 KO aECs were plated on collagen‐coated (10 μg/mL) 50 kPa PA hydrogels and then treated with or without 5 ng/mL of TGFβ1 for 30 min. Cells were transfected with Ad‐TRPV4‐WT (with or without GSK219), Ad‐TRPV4‐Δ1‐30, Ad‐TRPV4‐Δ1‐130, Ad‐TRPV4‐Δ100‐130, or Ad‐Vec. Immunoblotting assay and quantification of p‐MLC2 (Thr18/Ser19) relative to total MLC2 protein levels are shown. All experiments performed three times. (G) Bar graphs show quantification of results from D. Data analyzed using One‐way ANOVA followed by Bonferroni's multiple comparison test; ** p < 0.01, *** p < 0.001. (H) WT and TRPV4 KO aECs were treated with TGFβ1 (5 ng/mL) for 30 and 60 min or kept untreated (UT) for the control group. Immunoblotting assay of p‐Smad2, Smad2, p‐Smad3, Smad3, p‐AKT, AKT, and YAP/TAZ are shown. All experiments performed three times. (I–L) Bar graphs show quantification of results from H. Data analyzed using One‐way ANOVA; *** p < 0.001.

Article Snippet: For Western blot and immunofluorescence microscopy analyses, the following reagents and antibodies were used: Phospho‐MLC2 (p‐MLC2; 3674S), p‐Smad2, Smad2, p‐Smad3, Smad3, p‐AKT, AKT, YAP/TAZ, and MLC2 (3672S) antibodies were obtained from Cell Signaling Technologies (Danvers, MA), while the anti‐alpha smooth muscle actin (α‐SMA) antibody (A2547‐2ML) was purchased from Sigma (St. Louis, MO).

Techniques: Phospho-proteomics, Staining, Transfection, Quantitation Assay, Control, Western Blot, Comparison

Journal: The FASEB Journal

Article Title: TRPV4 ‐Mediated Mechanosensing Regulates the Endothelial‐to‐Mesenchymal Transition: Implications for Atherosclerosis

doi: 10.1096/fj.202403198R

Figure Lengend Snippet: Phospho‐MLC2 is linked to TRPV4‐mediated EndMT and traction force generation in aECs. (A) TRPV4 KO aECs were culture on stiff (50 kPa) PA hydrogels. Representative images from five different fields per condition showing morphological changes and co‐localization of F‐Actin (green) and ɑ‐SMA (red) with DAPI (blue) staining in TGFβ1 (5 ng/mL)‐stimulated TRPV4 KO aECs transfected with Ad‐TRPV4‐WT (with or without MLC2 inhibitor ML‐7) or Ad‐Vec. (B, C). Quantitation of results from Figure A. Data are expressed as mean ± SEM, n = 20 cells/condition; 1‐way ANOVA followed by Bonferroni test; *** p < 0.001, **** p < 0.0001. (D). Immunoblot analysis to confirm the ɑ‐SMA immunofluorescence data shown in panel A. (E) Bar graphs show quantification of results from D. Data analyzed using One‐way ANOVA; *** p < 0.001. (F) Immunoblot analysis to confirm that ML‐7 is attenuating p‐MLC2 levels. (G) Bar graphs show quantification of results from F. Data analyzed using t ‐test; *** p < 0.001. (H) TRPV4 KO aECs were plated on collagen‐coated (10 μg/mL) 25 kPa PA hydrogels containing 0.2 μm fluorescent beads in 24‐well plates with 5 ng/mL of TGFβ1 for 48 h. Cells were transfected with full‐length adeno‐TRPV4 (with or without MLC2 inhibitor ML‐7) or Ad‐Vec. Color‐coded traction vector maps indicate the magnitude of the traction vector, and corresponding phase images show the cells. (I) Quantitation of results from TFM assay as shown in Figure D. n = 10 cells/condition; *** p < 0.001, Student's t ‐test; RMS, root mean square.

Article Snippet: For Western blot and immunofluorescence microscopy analyses, the following reagents and antibodies were used: Phospho‐MLC2 (p‐MLC2; 3674S), p‐Smad2, Smad2, p‐Smad3, Smad3, p‐AKT, AKT, YAP/TAZ, and MLC2 (3672S) antibodies were obtained from Cell Signaling Technologies (Danvers, MA), while the anti‐alpha smooth muscle actin (α‐SMA) antibody (A2547‐2ML) was purchased from Sigma (St. Louis, MO).

Techniques: Staining, Transfection, Quantitation Assay, Western Blot, Immunofluorescence, Plasmid Preparation