Goat anti rabbit hrp

To detect the expression of ACE2 on the diltiazem-treated or CACNA1C-silenced cell surface, two 10-cm dishes of Vero-E6 cells were used to extract plasma membrane proteins by using the Minute Plasma Membrane Protein Isolation and Cell Fraction Kit (Invent Biotechnologies) following the manufacturer’s instructions. The total ACE2 in cells was extracted with RIPA buffer containing a protease inhibitor. Samples were incubated on ice for 30 min and centrifuged at 12,000 × g for 20 min at 4°C. Clarified cell lysate was diluted in denaturing SDS gel loading buffer and boiled for 15 min.
The samples were loaded onto a 4%–12% SDS-PAGE gel (Genscript) and separated by electrophoresis. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Merck-Millipore). The PVDF membrane was blocked with 5% skim milk and incubated with anti-Flag antibody (Genscript), anti-Myc antibody (Genscript), anti-ACE2 antibody (R&D system), anti-β-actin antibody (Zsbio), and anti-Zonula occludens protein 3 (ZO3) antibody (abcam). After being washed, the PVDF membrane was incubated with the following HRP-conjugated secondary antibodies: goat anti-rabbit HRP (Genscript), goat anti-mouse HRP (Genscript), and rabbit anti-goat HRP (Jackson ImmunoResearch). Signals were detected by using the enhanced chemiluminescence (ECL) reagent (Merck Millipore).

IHC was carried out as described previously [26 (link)]. The sections were deparaffinized, rehydrated and performed antigen retrieval with microwave method in 10 mM citrate buffer. The sections blocked with 3% H₂O₂ for 15 min, incubated with 5% normal goat serum in PBST for 1 h at 37 °C. Then sections were incubated with primary antibodies mouse anti-AdipoR2 (Santa Cruz; 1:50), rabbit anti-AdipoR1 (Abcam; 1:100), rabbit anti-Ki-67 (#9027, CST) and rabbit anti-CD31 (#77699, CST) at 4 °C overnight. After washing, followed by horseradish peroxidase-conjugated secondary antibody incubation for 1 h. Sections were incubated with developing solution (diaminobenzidine, DAB) and counterstained with hematoxylin (ZSGB-Bio, Beijing, China). Goat anti-mouse-HRP and goat anti-rabbit-HRP (Jackson ImmunoResearch) were used as the secondary antibodies.

Mouse anti-SCAR (DSHB P1C1-SCAR, RRID: AB_2618386) was used at a dilution 1:250. Rat anti HA-HRP (Roche catalog number: 12013819001, RRID:AB_390917) was used at a dilution of 1:500. Guinea Pig anti-Non-stop (Mohan et al., 2014a (link)) was used at a dilution of 1:1000. Guinea Pig anti-Ada2b (Kusch et al., 2003 (link)) was used at a dilution of 1:1000. Rabbit anti-Atxn7 (Mohan et al., 2014a (link)) was used at a concentration of 1:2000. Goat anti Guinea Pig HRP (Jackson ImmunoResearch INC catalog number: 106-035-003, RRID:AB_2337402) was used at a dilution of 1:10,000. Goat anti-mouse HRP (Jackson ImmunoResearch INC catalog number: 115-035-003, RRID:AB_10015289) was used at a dilution of 1:5000. Goat anti-Rabbit HRP (Jackson ImmunoResearch INC catalog number: 111-035-003, RRID:AB_2313567) was used at a dilution of 1:10,000.

Fat bodies (8–10 per sample) were dissected in ice-cold PBS and crushed in lysis buffer (2% SDS, 60 mM Tris-Cl, pH 6.8, and 1× PIC). Samples were cleared by centrifuging at 21,000 g for 30 min. Total protein was estimated by BCA assay (Pierce) and samples were boiled in 1× Laemmli buffer. Equal amounts of protein (30–40 μg/sample) were resolved on a 10% polyacrylamide gel and transferred onto a PVDF membrane (Immobilon-E, Merck). The membrane was blocked with 5% milk in TBS containing 0.1% Tween20 (TBS-T) for an hour followed by incubation with the primary antibody diluted in 5% milk in TBS-T. Following 3 washes with TBS-T, the membrane was incubated with the secondary antibody diluted in 5% milk in TBS-T for an hour, at room temperature. The membrane was washed thrice with 0.1% TBS-T, incubated with Immobilon Western Chemiluminescent HRP substrate (Merck), and visualized on a LAS4000 Fuji imaging system. The following antibodies were used: Rabbit anti-Dorsal, 1:5,000 (kind gift from the Courey laboratory); Mouse anti-Cactus, 1:100 (DSHB 3H12); Mouse anti-α-Tubulin, 1:10,000 (T6074, Sigma-Aldrich); Goat antirabbit HRP; and Goat antimouse HRP secondary antibodies, each at 1:10,000 (Jackson ImmunoResearch).

Cell lysates were electrophoresed on SDS-PAGE 4–20% gradient TGX stain-free gels (BioRad) and transferred to either 0.45 µm Immobilon-FL PVDF membrane (Millipore) or 0.2 µm nitrocellulose membrane (BioRad), followed by immunoblotting with indicated primary antibodies and secondary antibodies: anti-PAR (Trevigen), β-actin-horseradish peroxidase (HRP) (Santa Cruz), goat anti-rabbit HRP (Jackson Immunoresearch) and goat anti-mouse HRP (Jackson Immunoresearch). Chemiluminescent images of membranes were captured digitally using a Chemidoc MP Imaging System Touch (BioRad), with signals developed by Crescendo ECL reagent (Millipore) and quantified with the ImageLab 6.0.1 software (BioRad). Uncropped immunoblot images are shown in Supplemental Fig. 1.

Exosome samples solubilized in Laemmli buffer were loaded onto SDS-PAGE gels by equal volume or equal particle number[18 (link)], separated electrophoretically and transferred to nitrocellulose. Membranes were blocked with 10% fat-free milk/water for 30min at room temp on a rocking platform. Blocking solution was exchanged with primary antibody solutions (antibodies diluted in 5% fat-free milk/TBST) and incubated on a rocking platform for 4hrs at room temperature or overnight at 4°C. Primary antibodies used were as follows: anti-Fn (#sc8422, Santa Cruz), anti-annexin A2 (#610068, BD Transduction Laboratories), anti-annexin A6 (#ab52221, Abcam), lactadherin (MFG-E8)(#sc271574, Santa Cruz), myocilin (custom polyclonal [27 (link)]). Primary antibodies were removed and blots were washed 3 times, for 5 minutes with TBST. Antibody solutions containing secondary, HRP-conjugated antibodies (goat anti-mouse HRP # 115035146, goat anti-rabbit HRP # 111035144, Jackson ImmunoResearch, West Grove, PA) were added and incubated for 1hr at room temperature while rocking. Blots were then washed 3 times for 5 min in TBST and developed using chemiluminescent reagents (HyGLO; #E2400, Denville Scientific, South Plainfield, NJ) and X-ray film (#30–101, Genesee Scientific, San Diego, CA).