Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Splicing Shift of RAC1 Accelerates Tumorigenesis and Defines a Potent Therapeutic Target in Lung Cancer.

doi: 10.1002/advs.202503322

Figure Lengend Snippet: Figure 5. RAC1B preferentially interacts with GDS1 to stabilize EGFR. A) Scatter plot comparing the number of mass spectrums for candidate interacting proteins of RAC1B and RAC1A identified by FLAG antibody immunoprecipitation (IP) and mass spectrometry in PC9 cells stably expressing FLAG-RAC1B and RAC1A, respectively. B,C) Reciprocal co-immunoprecipitation and Western blot analyses in PC9 cells stably co-expressing FLAG-RAC1A or FLAG- RAC1B and HA-GDS1 or HA-GDIR. Cell lysates were immunoprecipitated with FLAG (B) and HA (C) antibodies respectively, and detected by Western blot with indicated antibodies. IgG antibody serves as the negative control in IP. D) Experimental scheme of RAC1 activation pulldown assay. E) Western blot analyses of PAK-GST pulldown products in PC9 cells co-expressing FLAG-RAC1A or FLAG-RAC1B and vector control, HA-GDIR1 or HA-GDS1. Loading control: 𝛽-actin. F) Western blot analysis of PAK-GST pulldown products in FLAG-RAC1B PC9 cells under control or GDS1 silencing. Loading control: 𝛽-actin. G) Colony formation of PC9 cells upon FLAG-RAC1B overexpression without or with GDS1 silencing. Quantification of the colony area was shown at the right part of this panel. Error bar: ±SD, n = 3 biological replicates. * P < 0.05, ns: not significant, two-way ANOVA with Dunnett’s multiple comparison test. H) Western blot analysis of indicated proteins in PC9 cells under conditions as described in (G). I,J) Western blot analysis of EGFR protein expression in control and GDS1-silenced PC9 cells treated with DMSO control, HCQ (50 μm, 24 h) or MG132 (10 μm, 24 h) (I) and with DMSO control, M𝛽CD (5 mm, 12 h) or Filipin (1 μg mL−1, 12 h) (J), respectively. Loading control: 𝛽-actin.

Article Snippet: RAC-GTP Pull-Down Assay: RAC1A and RAC1B activities were estimated by the ratio of their active GTP-bound forms to their total protein levels in PC9 cells using Rac1 Pulldown Activation Assay Kit (Cytoskeleton, Cat. No. BK035) following the manufactures’ manual.

Techniques: Immunoprecipitation, Mass Spectrometry, Stable Transfection, Expressing, Western Blot, Negative Control, Activation Assay, Plasmid Preparation, Control, Over Expression, Comparison

Journal: Biology direct

Article Title: Inhibition of RAC1 activator DOCK2 ameliorates cholestatic liver injury via regulating macrophage polarisation and hepatic stellate cell activation.

doi: 10.1186/s13062-025-00612-3

Figure Lengend Snippet: Fig. 5 DOCK2 activates RAC1 to participate in BDL-induced liver injury and its deficiency suppressed LPS-induced M1 macrophage polarisation. (A and B) Representative microphotographs of double IF staining of DOCK2 and RAC1 in 3d and 2w BDL livers. Magnification: 600 fold. Scale bar: 50 μm. (C and D) Representative microphotographs of IF staining of GTP-RAC1 in 3d and 2w BDL livers. The quantification of mean fluorescence intensity (MFI) is shown in the right panels. Magnification: 400 fold. Scale bar: 50 μm

Article Snippet: RAC1 activation was evaluated using the Rac1 Pulldown Activation Assay Biochem Kit (Cytoskeleton, Inc., CO, USA; Cat. #: BK035) following the manufacturer’s instructions.

Techniques: Staining, Fluorescence

Journal: Biology direct

Article Title: Inhibition of RAC1 activator DOCK2 ameliorates cholestatic liver injury via regulating macrophage polarisation and hepatic stellate cell activation.

doi: 10.1186/s13062-025-00612-3

Figure Lengend Snippet: Fig. 6 Knockdown of DOCK2 suppressed LPS-induced M1 polarisation of mouse macrophages. (A) Schematic illustration of experimental protocols. (B) Protein levels of DOCK2 6 h after 100 ng/mL LPS stimulation. (C) Relative mRNA level of DOCK2 in RAW264.7 cells 48 h after adenovirus transduction. (D) 48 h after adenovirus transduction, cells were stimulated with 100 ng/mL LPS. Representative microphotographs of IF staining of iNOS 6 h later. Magnifica tion: 400 fold. Scale bar: 50 μm. (E) Relative mRNA levels of M1 macrophage cytokines (IL-6, TNF-α, and iNOS). (F) Representative microphotographs of IF staining of GTP-RAC1 in cells. Magnification: 400 fold. Scale bar: 50 μm. The quantification of mean fluorescence intensity (MFI) is shown in the right panel. (G) The pull-down assay of GTP-RAC1 by PAK-PBD protein beads. (H) Protein levels of DOCK2 in cells

Article Snippet: RAC1 activation was evaluated using the Rac1 Pulldown Activation Assay Biochem Kit (Cytoskeleton, Inc., CO, USA; Cat. #: BK035) following the manufacturer’s instructions.

Techniques: Knockdown, Transduction, Staining, Fluorescence, Pull Down Assay

Journal: Biology direct

Article Title: Inhibition of RAC1 activator DOCK2 ameliorates cholestatic liver injury via regulating macrophage polarisation and hepatic stellate cell activation.

doi: 10.1186/s13062-025-00612-3

Figure Lengend Snippet: Fig. 7 Knockdown of DOCK2 inhibits TGF-β1-induced activation of HSC cells. (A) Schematic illustration of experimental protocols. (B) Protein levels of DOCK2 24 h after 10 ng/mL TGF-β1 stimulation. (C) Relative mRNA levels of DOCK2 in HSCs 48 h after adenovirus transduction. (D) 24 h after adenovirus transduction, cells were stimulated with 10 ng/mL TGF-β1. Representative microphotographs of IF staining of α-SMA 24 h later. Magnification: 400 fold. Scale bar: 50 μm. (E) Protein levels ofα-SMA in cells. (F) Representative microphotographs of IF staining of GTP-RAC1 in cells. Magnification: 400 fold. Scale bar: 50 μm. (G) The quantification of MFI. (H) The pull-down assay of GTP-RAC1 by PAK-PBD protein beads. (I) Protein levels of DOCK2 in cells. (J) Venn analysis showing the common differentially expressed genes (DEGs;|Log2FC|>1 and p < 0.05) between DOCK2-silenced M1 macrophages and myofibro blasts. (K) GO biological processes and KEGG pathways enriched for these common DEGs

Article Snippet: RAC1 activation was evaluated using the Rac1 Pulldown Activation Assay Biochem Kit (Cytoskeleton, Inc., CO, USA; Cat. #: BK035) following the manufacturer’s instructions.

Techniques: Knockdown, Activation Assay, Transduction, Staining, Pull Down Assay