Jagged1

These procedures were performed as previously described (Tu et al., 2019 (link)). Serial 10 μm-thick coronal sections of the rat brain after fixation and dehydration were immersed in primary antibodies: Jagged1 (1:100, Santa Cruz, United States), Notch1 (1:1,000, CST, United States), Hes1 (1:100, Santa Cruz, United States), and CD31 (1:200, Abcam, United Kingdom)/CD31 (1:200, Affinity Biosciences, OH, United States) overnight at 4°C, followed by fluorescein-conjugated secondary antibodies (1:1,000, Yishan, China), and observed with fluorescence microscope (Olympus, Japan).

All reagents for solutions, unless otherwise specified, were purchased from Fisher Scientific (Waltham, MA, United States). Primary antibodies and stains were as follows: Alexa Fluor 633 Hydrazide (approximates elastin staining), Notch3 (Santa Cruz and Abcam), Jagged1 (Santa Cruz), Notch1 (Abcam), Notch2 (Abcam), and Pai-1 (Abcam). Secondary antibodies were: donkey anti-goat, donkey anti-rabbit or donkey anti-mouse Alexa 488 or Alexa 555, all from Invitrogen. Transwells (polyester, 0.4 μm pore diameter for imaging and 1.0 μm pore for luciferase assay) were purchased from Corning.

Antibodies raised against the V-ATPase ‘a’ subunit isoforms a1 and a2 were generated in our laboratory. The mouse anti-a2V neutralizing antibody against 488–510 amino acids of trans-membrane region (Antibody clone 2C1) and rabbit anti-a2NTD against N terminal domain (Antibody Clone 470) were used as described previously [25 (link), 44 (link), 60 (link)]. Anti a1 antibody was raised in rabbit against the synthetic peptides from unique regions of a1 (amino acids 73–95; RKANIPIMDTGENPEVPFPRD) by Covance (USA) and anti a3 antibody was purchased from Abnova, USA. Notch1 (antibody clone EP1238Y) and organellar markers Rab5, Pan-Cadherin and Golph4 were from Abcam. For Western Blot we used cleaved Notch1 antibody Val1744 (Cell signaling, Danvers, MA), Jagged1 (Antibody clone H114, Santa-Cruz, CA), LC3B (Abcam). β-actin (antibody clone AC-74) was purchased from Sigma Aldrich and used as the loading control. For immunohistochemistry we used Notch-1(Santa-Cruz, antibody clone C-20), and anti-a2V. For flow cytometry we used Notch1-APC (Biolegend, San Diego, CA) and FITC conjugated anti-a2V (Covance, Princeton, NJ).

Proteins were extracted from long bones or cells and quantitated by a kit (Bio-Rad, Mississauga, Ontario, Canada). Protein samples of 30 μg were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblotting was carried out as described previously [25] (link) using antibodies against Runx2 (MBL International, Woburn, MA), peroxisome proliferator-activated receptor γ (Ppar-γ, E-8, Santa Cruz, CA, USA), PTHR (clone 3D1.1, Millipore), insulin-like growth factor 1 (IGF-1, clone Sm1.2, Millipore), Jagged1 (Santa Cruz, USA), activated Notch1 (Abcam, USA) and β-tubulin (Santa Cruz, CA, USA). Bands were visualized using enhanced chemiluminescence (ECL, Amersham) and quantitated by Scion Image Beta 4.02 (Scion Corporation, Bethesda, MD, USA).

Cells were washed once with phosphate-buffered saline (PBS), lysed using sample buffer containing 10% SDS and analyzed by SDS-PAGE. Membranes were imaged on a Li-Cor scanner, and processed using ImageJ. To examine the induction of JAG1 protein expression, BAC cells were stimulated with 12 nM NRG1 (cat# 396-HB, R&D Systems, Minneapolis, MN, USA) for 8 h and lysed: antibodies and their dilutions were used as follows: Tubulin (1:5000) (cat# T4026, Sigma-Aldrich, St. Louis, MO, USA), NRG1 (1:1000) (cat# sc-28,916, Santa Cruz Biotechnology, Dallas, TX, USA), Jagged 1 (1:1000) (cat# sc-6011, Santa Cruz Biotechnology).

Immortalized lens cells were authenticated by immunostaining with lens markers and confirmed to be free of mycoplasma contamination as previously described (Li et al., 2019 (link)). They were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and L-glutamine, 1% penicillin-streptomycin. After serum starved for 24 hr, cells were subject to FGF2 (50 ng/ml) stimulation, with or without Mek inhibitor U0126 (50 μM), PD0325901 (50 μM) or PI3K inhibitor LY (50 μM) for the indicated time periods, and harvested in CelLytic-M lysis buffer (Sigma) with proteinase inhibitor cocktail (Thermo fisher). Protein lysates were collected following centrifugation at 12,000 g for 10 min and resuspended in SDS buffer. Equal amounts of total protein were loaded for western blot analysis and visualized using an Odyssey SA scanner (LICOR Biosciences, Lincoln, NE). Antibodies used were Jagged1 (H-114, Santa Cruz Biotechnology), Notch1 (#4380, Cell signaling Technology), phospho-ERK1/2 (sc-7383, Santa Cruz Biotechnology), ERK (#4695, Cell signaling Technology).

Podocytes were washed with precooling saline twice followed by 500 μL of pyrolysis buffer (20 mmol/L Tris-HCl, 2.5 mmol/L EDTA, 10% glycerol, 0.1% SDS, 1% Triton X-100). The buffer was placed in an icebath for 40 minutes and centrifuged 12,000/minute for 20 minutes at 4°C. The supernate was discarded. The extracted protein, SDS polyacrylamide gel electrophoresis, was transferred to the polyvinylidene fluoride membrane. The membranes were blocked by 5% nonfat milk for 2 hours at 37°C and then incubated with primary antibodies at 4°C overnight. The primary polyclonal antibodies were as follows: Jagged 1 (Santa Cruz) ‘Notch 1‘NICD 1‘Bcl-2 (1:200, rabbit; Abcam, Cambridge, UK); Hes 1, Hey 1 (1:2000, rabbit; Abcam); Bax (1:3000, rabbit; Proteintech, CA, USA); β-arrestin-1 (1:2000, rabbit; Abcam); β-arrestin-2 (1:2000, rabbit; Abcam); β-actin (1:1000, rabbit; Bioss, Beijing, China). After being washed with Tween20-Tris buffer solution, horseradish peroxidase conjugated secondary antibody (1:5000, goat) was incubated for 1 hour at 37°C. Protein bands were visualized using an ECL kit (Thermo Scientific, CA, USA). β-actin was used as an internal reference for quantification and protein levels were quantified using Image-Pro Plus 6.0 software.