Rabbit anti mouse antibody against tgfβ1

Flow cytometry was performed to assess for cell‐bound and intracellular TGFβ1 protein expression (Sueblinvong et al., 2014). In short, PLFs from wild‐type and NLS‐Tg mice were treated with alcohol (60 mM). Cells were harvested 24 hours later, permeabilized, and stained at a 1:50 concentration with a rabbit anti‐mouse antibody against TGFβ1 (Abcam, Cambridge, MA) and at a 1:100 concentration with a goat anti‐rabbit‐Alexa 488 secondary antibody (Invitrogen, Carlsbad, CA). Cells were analyzed for TGFβ1 expression using a FACSCalibur (Becton Dickinson, Mountain View, CA). Flow cytometry data were analyzed using the FlowJo vX.0.7 software (Tree Star, San Carlos, CA).

Flow cytometry was performed to assess for cell-bound and intracellular TGFβ1 protein expression. After 16 hours of treatment, GolgiPlug (1 μl/ml; BD Bioscience, San Jose, CA) was added and at 24 hours after treatment, cells were harvested, permeabilized, and fixed (using CytoFix/CytoPerm; BD Bioscience) and stained with 1:50 rabbit anti-mouse antibody against TGFβ1 (Abcam, Cambridge, MA) and 1:100 goat anti-rabbit-alexa 488 secondary antibody (Invitrogen, Carlsbad, CA). Cells were fixed with 5% formalin then analyzed for TGFβ1 expression using a FACSCalibur (Becton Dickinson, Mountain View, CA). Flow cytometry data were analyzed using the FlowJo vX.0.7 software (Tree Star, San Carlos, CA). In parallel, conditioned media were collected, cells were separated by centrifugation, proteinase inhibitors were added, and media were frozen prior to analysis for secreted TGFβ1 by ELISA (R&D Systems, Minneapolis, MN).