Anti rhoa

Mice were anesthetized using sodium pentobarbital (60 mg/kg i.p.). The dorsal half of the 4–5th lumbar spinal cord was removed, immediately frozen in liquid nitrogen, and stored at −80 °C. Tissues were sonicated in ice-cold (4 °C) RIPA lysis buffer (Beyotime Institute of Biotechnology) containing a cocktail of protease and phosphatase inhibitors. After incubation on ice for 15 min, homogenates were centrifuged at 12,000 rpm for 20 min at 4 °C. Protein concentrations were measured using a bicinchoninic acid (BCA) Protein Assay Kit (Pierce). Protein samples were then denatured at 95 °C and separated by 8% SDS-PAGE. After transfer to PVDF membranes and blocking with 5% nonfat milk, the membranes were incubated overnight at 4 °C with anti-RhoA (1:500; Abcam), anti-ROCK2 (1:500; Abcam) or anti-β-actin (1:1000; Abcam) primary antibodies. The membranes were washed with wash buffer and incubated for 2 h with alkaline phosphatase-conjugated secondary antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature. The immune complexes were then detected using a nitro blue tetrazolium/5-bromo-4-c- hloro-3-indolyl phosphate assay kit (Sigma, St. Louis, MO). Western blots were analyzed using densitometry with Adobe Photoshop software (Adobe Systems Inc.).

Synthetic elastin peptides (VGVAPG, AGVPGLGVG, GRKRK and TAMRA-AGVPGLGVG) were purchased from Proteogenix. Mouse anti-MMP-2 and anti-uPA antibodies were from Calbiochem. Y27632, blebbistatin, U0126, PD150606, lactose, chondroitin sulphate, nifedipine and EDTA were from Sigma-Aldrich. EGCG was purchased from Enzo Life Sciences. Rabbit anti-Hsp90, anti-cleaved caspase-3, anti-integrin αV, anti-p-ERM, mouse anti-αvβ3 and anti-αvβ5 integrin antibodies were from Ozyme. Rabbit anti-RPSA, anti-MMP-14, anti-calpain1, anti-ROCK2, anti-myosin light chain kinase, mouse anti-RPSA, anti-RhoA, anti-ROCK1 and mouse IgM isotype control antibodies were purchased from Abcam. Rabbit anti-p-LIMK-and goat anti-cofilin and anti-actin were from Santa Cruz. Anti-integrin αvβ3 antibody was purchased from Millipore. Annexin-5 alexa fluor^® 568, CellTrace Calcein Red-Orange, AM and DiOC18³ were from Invitrogen.

The active form of RhoA, GTP-bound RhoA, in BSMs was measured by GTP-RhoA pull-down assay as described previously [19 (link)]. In brief, the isolated main bronchial tissues were equilibrated in oxygenated Krebs–Henseleit solution at 37 °C for 1 h. After the equilibration period, the tissues were stimulated with PGD₂ (10⁻⁵ M) or ACh (10⁻³ M) for 15 min, and were quickly frozen with liquid nitrogen. The tissues were then lysed in lysis buffer with the following composition (mM): HEPES 25.0 (pH 7.5), NaCl 150, IGEPAL CA-630 1%, MgCl₂ 10.0, EDTA 1.0, glycerol 10%, 1× protease inhibitor cocktail (Nakalai tesque, Kyoto, Japan), and 1× phosphatease inhibitor cocktail (Nakalai tesque). Active RhoA in tissue lysates (200 µg protein) was precipitated with 25 µg GST-tagged Rho binding domain (amino acids residues 7–89 of mouse rhotekin; Upstate, Lake Placid, NY, USA), which was expressed in Escherichia coli and bound to glutathione-agarose beads. The precipitates were washed three times in lysis buffer, and after adding the SDS loading buffer and boiling for 5 min, the bound proteins were resolved in 15% polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with rabbit polyclonal anti-RhoA (Abcam, Cambridge, UK) as primary antibodies.

Western blot analysis was carried out using standard methods. Total protein was extracted from the cultured cells or tumor tissues with lysis buffer. Protein concentration was determined by using a BCA method. The protein sample was then separated by 8-10% SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with primary antibodies overnight at 4°C. Membranes were probed with anti-RAB1B (1:1000), anti-Ras (1:5000), anti-Raf1 (1:1000), anti-Rac1 (1:500), anti-RhoA (1:5000), anti-CDC42 (1:1000), anti-β-actin (1:5000), and HRP conjugated anti-mouse (1:5000) and anti-rabbit (1:5000) (Abcam, Burlingame, CA).

NaHS was obtained from Sigma Chemical (St. Louis, USA); [γ-³²P] ATP was obtained from China Tongfu Co., Ltd (Beijing, China); PKG1, ATP, DT-2, and ACh were obtained from Sigma-Aldrich (St. Louis, MO, USA); LDH test kit was obtained from Nanjing Jiancheng Biotechnology (Nanjing, China); NSE Assay Kit was obtained from Jiangsu Meimian Industrial, Co., Ltd (Jiangsu); Fluo-8 AM, anti-ROCK₂ (catalog number ab125025), anti-Phospho-RhoA Ser188 (catalog number ab41435), and anti-RhoA (catalog number 187027) were purchased from Abcam (San Francisco, California, USA); and anti-β-actin (catalog number AF7018) and goat anti-rabbit IgG secondary antibody (catalog number S0001) were purchased from Affinity Biosciences (Changzhou, China).

Liver tissue protein was obtained from tissue lysates for western blotting. The protein levels were determined using a BCA assay kit (Tiangen, Beijing, China). Denatured proteins were separated on 10% Tris-glycine polyacrylamide gels by SDS-PAGE and transferred to PVDF membranes. The PVDF membranes were incubated overnight at 4 °C with anti-collagen I (Abcam, Cat. ab138492), anti-MMP1 (Abcam, Cat. ab137332), anti-α -SMA (Abcam, Cat. ab32575), anti-TIMP1 (Abcam, Cat. ab109125), anti-NOX4 (Abcam, Cat. ab109225), anti-RhoA (Abcam, Cat. ab187027), anti-ROCK1 (Abcam, Cat. ab45171), and anti-GAPDH (Abcam, Cat. ab8245). The corresponding membrane-bound antibodies were detected by a hypersensitive chemiluminescence detection reagent.