To analyse the cytoskeleton of the macrophages, the actin cytoskeleton was visualized. Macrophages were harvested after 5 days culture, reseeded on glass coverslips and then activated with IFNγ/LPS or IL-4 to induce M1 or M2 subsets. M0 macrophages were cultured in medium without stimuli. After 48 h, cells were left untreated or activated with CCL2 10 μM (Peprotech, Heerhugowaard, The Netherlands) for 10 minutes fixed with 4% paraformaldehyde in PBS for 30 minutes at 4°C, rinsed with PBS and permeabilized with 0.2% Triton X-100 in PBS for 5 minutes. The cells were rinsed with 10 mM Glycine, rinsed with PBS, and incubated with Cdc42 (1:500 Abcam, Cambridge, UK) for 1 h. The cells were rinsed with PBS prior to staining with the second antibody goat anti rabbit ALEXA 488 (1:400, Invitrogen) and rhodamine phalloidin (1:300, Sigma Aldrich) a high affinity, fluorescent filamentous actin probe for 1 h [9 (link)]. Cells were washed twice and the nuclei counterstained using Hoechst (1:5000, Sigma Aldrich) and washed again and mounted in mounting medium (88% hydrolysed polyvinyl alcohol, Dako, Heverlee, Belgium). Images were captured on a Leica DM6000 microscope.
Cdc42
Manufactured by Abcam
Sourced in United Kingdom, United States
Cdc42 is a small GTPase protein that plays a crucial role in regulating various cellular processes, including cell morphology, migration, and signaling. It acts as a molecular switch, cycling between an active GTP-bound state and an inactive GDP-bound state. Cdc42 is involved in the regulation of the actin cytoskeleton, which is essential for cell shape changes, adhesion, and movement.
Automatically generated - may contain errors
Lab products found in correlation
Collagen 1, by Abcam (2 mentions)
α sma, by Abcam (2 mentions)
β actin, by Abcam (2 mentions)
Hoechst, by Merck Group (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Rhodamine phalloidin, by Merck Group (1 mentions)
Ripa lysis, by Thermo Fisher Scientific (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Beta galactosidase, by Abcam (1 mentions)
Phalloidin, by Thermo Fisher Scientific (1 mentions)
Biotinylated goat anti rabbit secondary antibody, by Zymo Research (1 mentions)
Occludin, by Zymo Research (1 mentions)
Goat anti rabbit secondary antibody, by Beyotime (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Twist, by Abcam (1 mentions)
Iqgap3, by Abcam (1 mentions)
Kif2c, by Abcam (1 mentions)
Kif4a, by Abcam (1 mentions)
Snail, by Cell Signaling Technology (1 mentions)
14 protocols using cdc42
1
Visualizing Macrophage Cytoskeleton Dynamics
2
Cardiac Fibroblast Protein Expression
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The proteins of heart tissue or cardiac fibroblasts were prepared in RIPA lysis (Thermo fisher Scientific, USA). The following antibodies including CREG (Abcam), αSMA (Abcam), collagen-1 (Abcam), cleaved caspase 3 (Cell Signaling Technology, USA), Bax (Cell Signaling Technology, USA), proliferating cell nuclear antigen (PCNA, Abcam), CDC42 (Abcam), Rac1 (Abcam) and RhoA (Abcam), p21-activated kinase 1 (PAK1, Cell Signaling Technology), phosphorylated-PAK1 (Abcam) and insulin-like growth factor-2 receptor (IGF2R, Abcam) were used. β-actin was as the internal reference (Abcam).
3
Histopathological Analysis of Lung Tissue
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Histopathological analysis was performed as described before25 (link). Briefly, mice were sacrificed and lung tissues were inflated and fixed in PFA, embedded in paraffin or frozen in optimal cutting temperature compound (OCT) and sectioned for hematoxylin and eosin (H&E) staining. Immunohistochemical and immunofluorescence analyses were performed as described34 (link). Antibodies against the following proteins were used: KI-67 (Novocastra Laboratories Ltd), CDC42 (Abcam), ZO1 (33-9100, Zymed); PAR6 (sc-14405, Santa Cruz), Occludin (353197a Zymed), Phalloidin (R415 Invitrogen), CCSP (Santa Cruz), SP-C (AB3786, Chemicon), Beta Galactosidase (ab9631, Abcam), Biotinylated goat anti-rabbit secondary antibody (ZYMED company), Alexa Fluor 555 or 488 conjugated anti-mouse, rat or rabbit IgG secondary antibodies (Invitrogen).
4
Comprehensive Protein Expression Analysis
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For Western blot analysis, protein from each sample of fresh tissue or cells was lysed in lysis buffer. Equal amounts of protein were separated via gel electrophoresis, transferred onto a nitrocellulose membrane, and blotted with primary antibodies. The incubation antibodies include IQGAP3 (Ab88353; Abcam), Twist (Ab175430; Abcam), Snail (#3879s; Cell Signaling Technology), KIF2C (Ab205026; Abcam), KIF4A (Ab3815; Abcam), CDC42 (Ab155940; Abcam), proliferating cell nuclear antigen PCNA; #13110; Cell Signaling Technology), p53 (#9282; Cell Signaling Technology), matrix metallopeptidase 9 (MMP9) (Ab119906; Abcam), ERK1/2 (#4695; Cell Signaling Technology), p-ERK1/2 (#4376; Cell Signaling Technology), and glyceraldehyde 3-phosphate dehydrogenase GADPH; #5174; Cell Signaling Technology). Blots were then incubated with goat anti-mouse secondary antibody (cat. #A0208; Beyotime) or goat anti-rabbit secondary antibody (cat. #A0216; Beyotime). GADPH served as a loading control. Values under each blot are shown as the ratios of target protein/GADPH and represent the mean of three independent assays.
5
Western Blot Protein Analysis Protocol
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Cells were lysed in ice-cold RIPA Lysis and Extraction buffer (Thermo Fisher Scientific, Shanghai, China). Western blotting was performed according to a standard method21 (link). All the experiments were repeated thrice. All the results are from separate blots to avoid possible problems related to incomplete stripping.
All of the primary antibodies (Cdc42 and GAPDH) and the secondary antibodies (antirabbit IgG conjugated with horseradish peroxidase (HRP)) are commercially available from Abcam (Cambridge, MA, USA).
All of the primary antibodies (Cdc42 and GAPDH) and the secondary antibodies (antirabbit IgG conjugated with horseradish peroxidase (HRP)) are commercially available from Abcam (Cambridge, MA, USA).
6
Protein Extraction and Western Blot Analysis
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Total proteins from GC tissues and cells were extracted using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) containing the protease inhibitors and phosphatase inhibitors. Protein samples (20 μL) in each group were separated by SDS-PAGE before being transferred onto polyvinylidene fluoride (PVDF) membranes (Beyotime, Shanghai, China), which were then blocked by 5% non-fat milk followed by incubation overnight at 4°C with primary antibodies against ITGB1 (Cell Signaling Technology, Beverly, MA, USA; #34971), FAK (Cell Signaling Technology, Beverly, MA, USA; #3285), pY397-FAK (Cell Signaling Technology, Beverly, MA, USA; #3281), CDC42 (Abcam, Cambridge, MA, USA; #ab187643), PXN (Abcam, Cambridge, MA, USA; #ab32084), pY118-PXN (Abcam, Cambridge, MA, USA; #ab109547), AKT (Proteintech, Wuhan, China; #10176-2-AP), pS473-AKT (Proteintech, Wuhan, China; #66444-1-IG), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Dallas, TX, USA; #sc-47724) (Table S4 ). Subsequently, the membranes were incubated with horseradish peroxidase-labeled secondary antibody (Proteintech, Wuhan, China) at room temperature for 1 h. Protein signals were developed using the Enhanced Chemiluminescence Kit (Beyotime, Shanghai, China).
7
Western Blot Analysis of Apoptosis Markers
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Protein lysate containing 20 µg of proteins was separated on a Bolt 4–12% Bis-Tris Gel (NovexTM, ThermoFisher Scientific, Goteborg, Sweden) and transferred to a nitrocellulose membrane using the iBlot Gel Transfer Device (ThermoFisher Scientific, Goteborg, Sweden). Incubation with primary antibody (Cathepsin B (D1C7Y), Cell Signaling, Leiden, The Netherlands, Catalog no. 31718; Cathepsin D, BD Biosciences, San Jose, CA, USA, Catalog no. 610800; CDC42, Abcam, Cambridge, UK, Catalog no. ab155940; RhoA (67B89), Cell Signaling, Leiden, The Netherlands, Catalog no. 2117; PARP, Cell Signaling, Leiden, The Netherlands, Catalog no. 9542; Caspase 9, Bioss, Nordic BioSite, Stockholm, Sweden, Catalog no. bs-0049R; beta actin, Sigma-Aldrich, Stockholm, Sweden, Catalog no. A5441) diluted in TBST containing 3.5% bovine serum albumin (BSA) was done overnight at 4 °C. Secondary antibodies, goat anti-rabbit IgG HRP (Southern Biotech, Stockholm, Sweden Catalog no. 4030-05), or goat anti mouse IgG HRP (Southern Biotech, Stockholm, Sweden Catalog no. 1030-05) were incubated for 1 h. Membranes were developed using the AmershamTM ECLTM Start Western Blotting Detection Reagent (GE Healthcare Life Sciences, Logan, UT, USA) and bands were visualized using the Bio-Rad Quantity One imaging system (Bio-Rad, Stockholm, Sweden). Images were quantified using ImageJ software.
8
Immunofluorescent Analysis of Cardiac Cells
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Heart tissues or cardiac fibroblasts were fixed with 4% paraformaldehyde, incubated with the primary antibodies against CREG (Sigma Aldrich, USA), CDC42 (Abcam), αSMA (Abcam), collagen-1 (Abcam), and PCNA (Abcam) for overnight, and incubated with secondary antibodies (Thermo fisher Scientific). Cell nuclei were stained with DAPI. Samples were scanned using confocal microscopy (Zeiss).
9
Activation Assays for Cdc42, Rac1, RhoA, and AKT
10
Immunohistochemical Analysis of Skin Markers
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Primary antibodies against the following antigens were used: K1, K14, K15, K19, E-cadherin, Cdc42, AP-1, p-PAK1 (Abcam, UK), K6, loricrin, involucrin (Covance, USA), β-catenin, bromodeoxyuridine (BrdU; Santa Cruz, USA), α6-integrin (BD Biotech, USA), F4/80 (eBioscience, USA), SPRR1A (Biorbyt, UK), SPRR1B (Aviva Systems Biology, USA), desmoplakin, ZO-1, SPRR2D, PAK1, cyclin D1, caspase 3, occludin and SPRR2G (Proteintech, China), Bim, p-Bim, Bcl-2, p-Bcl-2, JNK, p-JNK, p38, p-p38, ERK, and p-ERK (Cell Signaling, USA).
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