Affinipure goat anti rabbit hrp igg

The extent of polymerization of globular (G‐) to filamentous (F‐) actin in ASM strips was evaluated using a G‐/F‐actin in vivo assay kit (Cytoskeleton Inc., Denver, CO) as previously described (Jones et al., 1999b; Dogan et al., 2017). From each trachea, 32 ASM strips were dissected (four TNFα‐treated and 4 control × 4 ACh concentrations) to evaluate the effect of ACH stimulation on actin polymerization. The ASM strips were exposed to 0 μmol/L, 1 μmol/L, the EC₅₀ concentration for each condition (1.3 μmol/L for TNFα and 2.6 μmol/L for control) and 10 μmol/L for 2 min and then flash‐frozen in liquid nitrogen. Subsequently, the ASM strips were thawed at room temperature and then minced in F‐actin stabilization buffer (composition in mmol/L; 50 PIPES pH 6.9, 50 KCl, 5 MgCl₂, 5 EGTA, 5% [v/v] glycerol, 0.1% Nonidet P40, 0.1% Triton X‐100, 0.1% Tween 20, and 0.1 % 2‐mercaptoethanol, supplemented with 1 mmol/L ATP and 1% protease inhibitor cocktail). Separation of F‐ and G‐actin was performed using standard western blotting technique with incubations of primary rabbit polyclonal anti‐actin antibody (1:1000 dilution) (Cytoskeleton Cat# AAN01, RRID:AB_10708070) and secondary peroxidase AffiniPure goat anti‐rabbit HRP IgG (1:10 000 dilution) (Jackson ImmunoResearch Labs Cat# 111‐035‐144, RRID:AB_2307391). The actin bands were analyzed with a Kodak Image System (Kodak Inc., CA, USA).

Actin polymerization in porcine ASM was determined as described previously (Dogan et al., 2017; Jones, Perkins, et al., 1999; Sieck et al., 2019; Tang & Gunst, 2004) using a G‐/F‐actin in vivo assay kit (Cytoskeleton Inc., Denver, CO). Briefly, one pair of permeabilized ASM strips were treated with 1 µM Cyto‐D or left untreated in pCa 9.0 for 10 min at 22°C and another pair of permeabilized ASM strips were treated with 1 µM Cyto‐D or left untreated in pCa 9.0 for 10 min then stimulated with pCa 4.0 solutions for 10 min at 22°C. The ASM strips (4 per trachea) were then snap‐frozen. Subsequently, the ASM strips were thawed at room temperature and then minced in F‐actin stabilization buffer (composition in mM; 50 PIPES pH 6.9, 50 KCl, 5 MgCl₂, 5 EGTA, 5% (v/v) Glycerol, 0.1% Nonidet P40, 0.1% Triton X‐100, 0.1% Tween 20, and 0.1% 2‐mercapto‐ethanol, supplemented with 1mM ATP and 1% protease inhibitor cocktail). Separation of F‐ and G‐actin was performed using standard western blotting technique with incubations of primary rabbit polyclonal anti‐actin antibody (1:1,000 dilution) (Cytoskeleton Cat# AAN01, RRID:AB_10708070) and secondary peroxidase AffiniPure goat anti‐rabbit HRP IgG (1:10,000 dilution) (Jackson ImmunoResearch Labs Cat# 111–035–144, RRID:AB_2307391). The actin bands were analyzed with a ChemiDoc MP Imaging System (Bio‐Rad Laboratories, Hercules, California, U.S.).

The sources of antibodies and the dilutions used in experiments were as follows: rabbit anti-β-PIX (Millipore 07-1450, Darmstadt, Germany; dilution for Western blotting: 1/1000; dilution for immunofluorescence: 1/500); mouse anti-paxillin (BD Bioscience 610052, San Jose, California, USA; dilution for immunofluorescence: 1/1000); mouse anti-GAPDH (GeneTex GTX627408, Hsinchu, Taiwan; dilution for Western blotting: 1/1000); DNase I (Sigma-Aldrich SAB2702031, Saint Louis, Missouri, USA; dilution for immunofluorescence: 1:1000); Alexa Fuor 488-antimouse IgG (Jackson ImmunoResearch 715-546-151/J1, West Grove, Philadelphia, USA; dilution for immunofluorescence: 1/300); Alexa Fuor 568-antimouse IgG (Invitrogen A11031; dilution for immunofluorescence: 1/300); Alexa Fuor 568-anti-rabbit IgG (Invitrogen A11036; dilution for immunofluorescence: 1/300); Alexa Fuor 647-antimouse IgG (Jackson ImmunoResearch 715-606-151/J1; dilution for immunofluorescence: 1/300). Alexa Fluor 488 phalloidin (A12379; dilution for immunofluorescence: 1/400) and Alexa Fluor 568 phalloidin (A12380; dilution for immunofluorescence: 1/400) were obtained from Invitrogen. HRP-AffiniPure goat antimouse IgG (115-035-174; dilution for Western blotting: 1/5000) and HRP-AffiniPure goat anti-rabbit IgG (211-032-171; dilution for Western blotting: 1/5000) were obtained from Jackson ImmunoResearch.