Mab1835

To inhibit TGFβ receptor signaling, the ALK4/5/7-kinase inhibitor SB-505124 (Sigma) was used in a concentration of 5 μM. DMSO was used as solvent control. Cells were pre-incubated with the inhibitor for 1 h before use. To neutralize TGFβ1, TGFβ2, and TGFβ3, a monoclonal mouse IgG1 antibody (MAB1835, R&D systems) was used in a concentration of 5 μg/ml. Mouse IgG1 was used as the isotype control.

Kidneys were embedded in OCT (4583, SAKURATissue-Tek®, Torrance, CA, USA) on dry ice. Five-micrometer sections were cut and performed with immunostaining. Briefly, the slices were fixed in 10% neutral buffered formalin; then washed with PBS and treated with 0.2% Triton X-100; mouse anti-αSMA (MS-113-P, Thermofisher, USA) and rabbit anti-synapotodin (sc-50459; Santa Cruz Biotechnology, Santa Cruz, CA, USA); or mouse anti-collagen 1α (ab6308, abcam, USA) and rabbit anti-synapotodin; or mouse anti-fibronectin (sc-271098, Santa Cruz, USA) and rabbit anti-synapotodin; or mouse anti-TGFβ (MAB1835, R&D, USA) and rabbit anti-synapotodin were incubated with the slices after blocked with 1% BSA; Donkey anti-mouse IgG-488 (ab150105; Abcam, Shanghai, China) and donkey anti-rabbit IgG-647 (ab150075; Abcam) were incubated with the slices; Hoechst 33342 was then used to stain the nucleus; the slices were mounted in VECTASHIELD Mounting Medium (H-1000, Vector Laboratories, Inc., Burlingame, CA, USA) staining. Images were obtained using a confocal microscope (TCS-SP8; Leica, Buffalo Grove, IL, USA). Positive cells with both synapotodin and EMT markers located in the glomeruli were counted and divided by synapotodin-positive cells located in the glomeruli. At least 100 glomeruli were analyzed in each group.

We performed intraperitoneal injections of TGF-β neutralizing-antibody (TNA; TGFb1,2,3—MAB1835 from R&D Systems) or control solution (TGF-β vehicle) into opossum pups every day from P16 to P22 [33 (link)]. The antibody was administered at 10 ng kg⁻¹. Pups were euthanized on postnatal day 22. To confirm the knockdown of TGF-β signalling, we cryosectioned the middle ear regions of TNA and control pups at P22, and performed IF for anti-p-Smad2 (Cell Signaling Technology) [34 (link)]. pSMAD is a downstream protein of TGF-β signalling [34 (link)–36 (link)]. MC morphology was visualized using micro-CT scanning and clearing and staining [34 (link)], and apoptotic cells (Cell Signaling Technology) using TUNEL on cryosectioned middle ear sections. The length and width of the skull and ectotympanic was measured in triplicate and averaged for each micro-CT scanned TNA and control pup, and statistically compared using the Wilcoxon–Mann–Whitney rank sum test [26 (link)]. For details, see the electronic supplementary Materials and Methods.

Hippocampal neurons were isolated from C57Bl6 mice at embryonic day (E)18.5 of gestation, as described previously (Lacmann et al., 2007 (link)). Cultures were treated with human recombinant TGF-β2 (2 ng/ml; R&D Systems) for 5, 10, 15, 30 and 60 min or with a pan-TGF-β antibody (a function-blocking anti-TGF-β1, -β2 and -β3 antibody; 10 µg/ml; cat. no. MAB1835, R&D Systems). At day in vitro (DIV)12, or DIV18 cells were processed for RT-PCR, immunoblotting or immunocytochemistry.

A375 or A375-EmGFP-ITGB1 cells were plated in triplicate in 96 wells (1.2 x 10⁴ cells per well) in full serum conditions and allowed to adhere for 24 hours. Cells were replaced with serum free medium and if indicated treated with IgG ([3.3 μg/ml] R&D systems MAB1835) or α-Integrin β1 activating antibody TS2/16.2.1 (TS2/16 ([10 μg/ml] ATCC clone HB243) for 20 hours. Supernatant was collected and tMLEC reporter cells were then incubated for 16–20 hours with the supernatant. 1D11 α-TGF-β1,2,3 antibody 1D11.16.8 ([4 μg/ml] BioXCell) was added to the supernatant of some samples as a control. After incubation a luciferase assay was performed according to manufacturers instructions (Promega, luciferase Assay System, E1500).