Chemidoc analyzer

Intestinal tissues were homogenized in Radioimmunoprecipitation (RIPA) (150 mM NaCl/1.0% Triton X-100/0.5% sodium deoxycholate/0.1% SDS/50 mM Tris; pH 8.0) lysis buffer containing Complete Ultra protease inhibitors (Roche, Indianapolis, IN, USA). Protein concentrations were determined using Pierce BCA protein assay kits (ThermoFisher Scientific) and normalized amounts of protein were denatured and reduced with LDS/DTT buffer (Invitrogen). Five–ten μg of protein was resolved in NuPAGE 4–12% Bis-Tris gels run in a 3-morpholimopropane-1-sulfonic acid (MOPS) buffer (Invitrogen), transferred onto nitrocellulose membranes (Invitrogen), and blocked with 5% non-fat dry milk(NFDM) in Tris-bufferd saline, 0.1% Tween 20 (TBST). EpCAM, TROP2, and claudins were detected with relevant primary antibodies (see above) after overnight incubation at 4 °C. TBST-washed blots were incubated for 1 h at RT with horseradish peroxidase-conjugated secondary Ab (Jackson ImmunoResearch, West Grove, PA, USA), washed, and exposed to SuperSignal West Pico chemiluminescent substrate or SuperSignal West Dura (Thermo Fisher) extended duration substrate. Proteins of interest were detected with X-ray film using a Kodak 2000A film processor. Blots were digitalized using Biorad’s Chemidoc analyzer and Quantity One 4.4.1 software.

Indicated cells post-manipulation were washed twice with cold PBS and lysed with cold 1% NP-40 lysis buffer supplemented with 1X protease inhibitor cocktail (Roche Applied Biosciences) for 15 mins at 4 °C. The cell lysate was centrifuged at 18,928 × g for 15 min. This was followed by protein estimation using Bradford reagent. Subsequently, 40–50 µl of total protein was mixed with an equal volume of 2X Laemmli sample buffer and heated at 95 °C for 5 mins. This was followed by resolution with SDS–PAGE gel. The resolved proteins were transferred onto nitrocellulose membrane and blocked in 5% skimmed milk reconstituted in 1X PBS containing 0.1% Tween-20, and probed overnight with the respective primary antibody. The membrane was then washed with 1× PBS supplemented with 0.1% Tween-20; three times at 15 min intervals. This was followed by 1 h incubation in conjugated horseradish peroxidase HRP secondary antibody (Santa Cruz Biotechnology). Post-incubation, the blot was again washed three times as above and then overlaid with enhanced chemiluminescence (ECL) substrate (Pierce/Bio-Rad) and visualized on X-ray film by image processor or digitally by Chemidoc analyzer (Bio-Rad) and analyzed by Image Lab 6.0.1 software. The density of the various bands was quantified using the Image-J 1.52e software.

Protein samples were solubilized in SDS sample buffer, separated on SDS-PAGE and electro transferred onto polyvinylidene fluoride membranes. Antibodies were added, and the protein-antibody complexes were labeled using a chemiluminescence reagent (Luminata Crescendo Western HRP Substrate; Merck kGaA). The signals were detected with ChemiDoc analyzer (Bio-Rad Laboratories). The antiserum against FZL was produced by immunizing rabbits with a purified recombinant FZL (60-475 aa) protein (PhytoAB INC). Specific antibodies against CURT1A and RbcL were kindly provided by Chanhong Kim (CAS Center for Excellence in Molecular Plant Science) and Hiroshi Shimada (Hiroshima University), respectively. The antibodies against Tic110, D1 and VIPP1 were described previously (Zhang et al., 2012 (link); Kato and Sakamoto, 2014 (link)) For detection of photosynthetic proteins PsaA, PetA, CF₁β, CF₁γ, PsbS, Lhcb1 and Lhca1, commercially available polyclonal antibodies were used (Agrisera).

Granulosa cells were collected in Laemmli buffer (Bio-rad) containing DTT (Omnipur), phosphatase and protease inhibitors (G Biosciences), and were boiled at 95°C for five minutes. Protein extracts were resolved by polyacrylamide electrophoresis and transferred to nitrocellulose membranes. After blocking with 5% milk in TBS-T, the membranes were incubated overnight at 4°C with primary antibodies (1:1000) followed by washing with TBS-T (3X 10 min each) and incubation with secondary antibody (1:10000) for 1hour at room temperature. The immunoblotted proteins were detected using Immun-Star Kit and Chemidoc Analyzer (BioRad). Whenever necessary, the membranes blotted with one primary antibody were stripped using stripping buffer (10% SDS, 0.5 M Tris-Hcl, DEPC H2O ml and 2-mercaptoethanol) and re-blotted with another antibody. Antibodies used: MAPK3/1 (#4695), phoshpho-MAPK3/1 (Thr202/Tyr204)(#4376) from Cell Signaling; EGR-1 (#sc-189x) form Santa cruz biotechnology; beta actin (ab8227) and Goat anti-rabbit-IgG (ab6721) from Abcam.

Whole-cell extracts were prepared in cell lysis buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, protease inhibiter [Roche]) with sonication (30 s ON/90 s OFF × 6 times) using Bioruptor (Cosmobio). Protein samples were separated by SDS-PAGE and transferred onto a PVDF membrane using a semi-dry transfer system (Trans-Blot Turbo, BioRad). The transferred membrane was blocked with TBS-T (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.05% Tween20) containing 5% BSA. The membrane was incubated 1 hour in a primary antibody diluted with Can Get Signal solution 1 (TOYOBO), and washed with TBS-T three times. The membrane was then incubated for one hour in a secondary antibody diluted with Can Get Signal solution 2 (TOYOBO). Washing three times with TBS-T, signals were developed with ECL prime western blotting detection reagents (GE Healthcare), and subsequently detected using the Chemidoc analyzer (BioRad). Primary antibodies were used at the following dilution rate. Anti-actin antibody (Thermo Fisher Scientific, MS-1295-P1): 1/1000. Anti-H3 antibody (Abcam, ab1791): 1/10,000. Anti-GFP antibody (MBL, 598): 1/5000. Anti-rabbit IgG (Promega, W4011) or anti-mouse IgG (Promega, W4021) were used as secondary antibodies at 1/10,000 dilution.