Goat anti rabbit hrp linked

The secondary antibodies used were as follows: goat anti-rabbit HRP-linked from Cell Signaling Technology; donkey anti-mouse Alexa488-linked and donkey anti-mouse Alexa594-linked from Invitrogen; and anti-mouse IgG MicroBeads from Miltenyi Biotech.

Mouse anti-human beta-actin, rapamycin and doxycycline hyclate were from Sigma. Helenalin, SN50 and Compound C were from Merck Biochemicals. Caffeic acid phenethyl ester (CAPE) was from Tocris (R&D Biosystems). Mouse anti-human MMP-8, mouse anti-human MMP-9, rabbit anti-human GAPDH, rabbit anti-human histone 2B, rabbit anti-human phospho-p70S6k (T229), rabbit anti-Mycobacterium tuberculosis, rabbit anti-human neutrophil elastase, rabbit anti-human phospho-AMPK alpha 1 and 2 (T172), sheep anti-human histone 2B, rabbit anti-human histone H3 (citrulline 2 + 8 + 17), donkey anti-Sheep IgG DyLight 488, goat anti-mouse DyLight 549, goat anti-rabbit IgG Cy5 were from Abcam. Rabbit anti-human phospho-Akt, total-Akt, phospho-AMPKα1/2 (T172), total AMPKα, and goat anti-rabbit HRP linked were from Cell Signalling Technology. Goat anti-mouse IgG (H+L) was from Jackson Immunoresearch laboratories. Goat anti-human MMP-8 was from R&D Biosystems and mouse anti-human MMP-9 was from Millipore. Rabbit anti-human MMP-8 was from Novus Biologicals. Mouse anti-human neutrophil elastase was from Dako. mouse anti-human MMP-9 was from Millipore.

PHBF cells were plated at 100,000 cells/well in a 24-well cell culture plate overnight, serum-starved for 24 h and then cultured in serum-free DMEM with the indicated concentration of TGFβ1 with or without Galunisertib for 30 min. Cells were lysed in protein lysis buffer containing T-PER tissue protein extraction reagent (ThermoFisher, #78510), cOmplete™ Protease Inhibitor Cocktail (Sigma-Aldrich, #11697498001) and PhosSTOP™ phosphatase inhibitor cocktails (Sigma-Aldrich, #4906845001). Total protein was diluted and normalized to 0.5 µg/µL with 4× LDS sample buffer (ThermoFisher, #84788). In total, 10 µg of total protein was loaded into each well of a NuPAGE 4–12% Bis-Tris Midi Gel (Invitrogen), followed by protein transfer from the gel to the membrane using Trans-Blot Turbo (Bio-Rad). The Phospho-Smad2 was first revealed following the general protocol western blot from Bio-Rad. Briefly, the membrane was blocked for 1 h, incubated with Phospho-Smad2 antibodies overnight at 4 °C (Ser456/467, 1:200, Cell Signaling #3108, clone 138D4), washed and incubated with secondary antibodies goat anti-rabbit HRP-linked (Cell Signaling, #7074, dilution: 1:15,000). To analyse the total Smad2/3, the membrane was stripped and then incubated with Smad2/3 antibodies (Cell Signaling # 8685, clone D7G7, dilution 1:1000).