Ab96698

Subsequent to isolation from tissues or cells using lysis buffer encompassing protease inhibitor cocktail, the total protein was then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes. Subsequent to 1-h membrane blocking with 5% skimmed milk powder at ambient temperature, overnight membrane incubation was implemented with primary rabbit antibodies against SUV39H1 (A3277, abclonal, 1: 2,000), Smad9 (ab96698, 1: 1,000, Abcam), BMP4 (A11315, abclonal, 1: 2,000), phosphorylated (p)-Smad9 (#13820, 1: 1,000, Cell Signaling Technologies, Beverly, MA, USA), and GAPDH (AC033, abclonal, 1: 50,000) at 4 ℃. The next day, the membranes were re-probed with horseradish peroxidase-conjugated secondary antibodies against rabbit immunoglobulin G (IgG) (AS014, abclonal, 1:10,000) or mouse IgG (A3950, abclonal, 1:10,000) at ambient temperature for 1 h. Subsequent to development using enhanced chemiluminescence reagents (Thermo Fisher Scientific), images were obtained using a ChemiDox XRS Plus luminescent image analyzer (Bio-Rad Laboratories, Hercules, CA, USA). The obtained images were then analyzed using Image-Pro Plus 6.0 software (three repeated experiments). GAPDH was used as a loading control.

Developmentally staged larvae (after euthanisation in MS222) were fixed in 4% paraformaldehyde (1 h), dehydrated to 100% methanol, and stored at −20 °C before staining. Immunolabeling was done as previously described [21 (link)]. Primary antibodies were anti-Smad9 (ab96698, Abcam, Cambridge, MA, USA) used at a 1:200 dilution, anti-Col2a1 (II-II6B3, DSHB, University of Iowa, Iowa, IA, USA) used at 1:20 and anti-GFP (chicken polyclonal, Abcam, ab13970) used at a 1:300 dilution in blocking buffer (5% horse serum). Additionally, rabbit anti-Smad1 (1:100 dilution, sc-6031-R, Santa Cruz, Dallas, TX, USA), rabbit anti-Smad5 (1:200 dilution, ab227090, Abcam), and rat anti-mCherry [16D7] (1:100 dilution, M11217, Invitrogen, Carlsbad, CA, USA). Primary antibodies were used in 5% horse serum in 0.2% PBS-Triton-X100 blocking buffer. Secondary antibodies Alexa Fluor 488 anti-rabbit, Alexa Fluor 568 anti-chicken, Alexa Fluor 647 anti-mouse (A21206, A11041, and A31571 respectively, Invitrogen, Carlsbad, CA, USA), and Dylight 650 anti-Rat (SA5-10029, ThermoFisher, Waltham, MA, USA) were used at a 1:500 dilution. Samples were mounted in 1% low melting point agarose and imaged as described below.

BMPre:GFP (Tg(5xBMPRE‐Xla.Id3:GFP))27 and sp7:GFP (Tg(Ola.sp7:NLS‐GFP))28 transgenic fish (in London AB background) were housed and maintained in standard conditions.29, 30 Experiments were approved by the University of Bristol Animal Welfare and Ethical Review Body (AWERB) and performed in accordance with a UK Home Office project license. Developmentally staged larvae (after euthanization in MS222) were fixed in 4% paraformaldehyde (1 hour), dehydrated to 100% methanol, and stored at −20°C before staining. Immunolabeling was as previously described.31 Primary antibodies were anti‐Smad9 (rabbit polyclonal, Abcam, Cambridge, MA, USA, ab96698) used at a 1/100 dilution and anti‐GFP (chicken polyclonal, Abcam, ab13970) used at a 1/200 dilution in blocking buffer (5% horse serum). Secondary antibodies were used (A21206 and A11041, Invitrogen, Carlsbad, CA, USA) in a 1/400 dilution and samples incubated with DAPI (Sigma‐Aldrich, St. Louis, MO, USA, 1/1000 dilution) to visualize nuclei. Samples were mounted in 1% low melting point agarose and imaged with a confocal laser scanning microscope (Leica, Buffalo Grove, IL, USA, SP5II AOBS attached to a Leica DM I6000 inverted epifluorescence microscope) using a 40× PL APO CS (1.3 numerical aperture) lens. Images were processed and color balanced in Fiji.32