Ab96587

The sections of the small intestine and colon were incubated with specific primary anti-IgA antibody (ab223410, Abcam, Cambridge, MA, USA) and anti-ZO-1 antibody (ab96587, Abcam, Cambridge, MA, USA), respectively, overnight at 4°C. Subsequently, the sections were washed 3 times with 1× PBS and incubated with fluorochrome-conjugated secondary antibodies in the dark for 1 h at 37°C. Finally, DAPI (4, 6-diamidino-2-phenylindole, blue, Southern Biotech) was added to the sections to dye the nucleus. Fluorescence photographs were observed using a fluorescence microscope DM5000 B (Leica, Germany).

HUVECs were seeded on 12‐ or 6‐well plates at a density of 40 000 or 200 000 cells·well⁻¹, respectively. After 24 h, HUVECs were synchronized with EBM™‐2 supplemented with 2% FBS for 24 h. Cells were then lysed with RIPA buffer (ELPIS‐BIOCH, Daejeon, Korea) containing phosphatase inhibitor cocktail 3 (Sigma, MO, USA) at 5 min or the indicated time points after VEGF treatment. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, MA), and an equal concentration of protein was loaded for western blotting analysis. The following primary antibodies were used in this study: mTOR (#2983, Cell Signaling, Danvers, MA), p‐PKCα S657 (ab180848, Abcam, Boston, MA), PKCα (sc‐8393, Santa Cruz, Santa Cruz, CA), p‐S6 S235 (ab12864, Abcam), S6 (sc‐74459, Santa Cruz), β‐actin (sc‐47778, Santa Cruz), and ZO‐1 (ab96587, Abcam). The primary antibodies were then bound to peroxidase‐conjugated secondary antibodies diluted in blocking solution, and their binding on the membrane was detected using enhanced chemiluminescence (Luminograph II, ATTO, Tokyo, Japan).

The following primary antibodies were employed (dilution): ZO1 (1:1000, ab96587, Abcam, USA), occludin (1:1000, ab167161, Abcam, USA), claudin-1 (1:1000, ab15098, Abcam, USA), IκBα (1:2000, 4812, Cell Signaling Technology, USA), p-IκBα (1:2000, 2859, Cell Signaling Technology, USA), NF-κB p65 (1:1000, ab16502, Abcam, USA), monocyte chemotactic protein-1 (MCP-1, 1:1000, ab7202; Abcam, USA), cyclooxygenase-2 (COX-2, 1:1000, ab62331; Abcam, USA), Keap1 (1:1000, ab139729, Abcam, USA), Nrf2 (1:1000, ab31163, Abcam, USA), heme oxygenase 1 (HO-1, 1:2000, ab68477, Abcam, USA), catalase (1:1000, ab52477, Abcam, USA), NAD(P)H quinone dehydrogenase 1 (NQO1, 1:1000, ab28947, Abcam, USA), α smooth muscle actin (α-SMA, 1:300, ab7817, Abcam, USA), collagen I (1:5000, ab34710, Abcam, USA), and fibronectin (1:1000, ab2413, Abcam, USA). Western blot analysis was performed as previously described^{24 (link),26 (link),27 (link)}. Blots were obtained with ECL reagent and protein concentrations were normalized by actin expression. Specific bands indicating target proteins were analyzed using ImageJ 1.48 v software.

Cross-sections (10 μm thickness) of rat colon were cut with a cryostat (Leica CM3050S, Nussloch, Germany). Colon cryosection were fixed with an acetone:methanol mixture (1:1) at 20 °C for 2 min and then rehydrated in phosphate-buffered saline (PBS) (3 × 5 min). After washing, sections were permeabilized with 0.5% Triton X-100 in PBS for 15 min and blocked for 40 min with 4% nonfat milk in 20 mM Tris, pH 7.2, and 150 mM NaCl. The sections were incubated with primary antibodies: rabbit polyclonal anti-ZO-1 (ab96587, Abcam, Cambridge, MA, USA) and mouse monoclonal anti-occludin (OC-3F10, 33-1500, Life Tecnologies, Carlsbad, CA, USA) in PBS containing 1% BSA overnight at 4 °C. After rinsing with PBS (3 × 5 min), the sections were incubated with the secondary fluorescent antibody Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 568-conjugated donkey anti-mouse IgG (1:200; Molecular Probes, Life Technologies, Paisley, UK) and 4′,6-diamidino-2-phenylindole (DAPI, nuclei dye), for 1 h at room temperature. After incubation, the sections were washed with PBS (3 × 5 min), and the slides were mounted using the Glycergel mouting medium (Dako, Carpinteria, CA, USA). Anti-ZO-1 and anti-occludin immunostaining samples were imaged using a confocal fluorescence microscope (LSM 710, Carl Zeiss, Gottingen, Germany).

Western blot analysis was done as described previously.¹⁴ The antibodies used in IHC were also applied to the Western blotting analysis. Primary antibodies were raised against proteins associated with spermatogonia proliferation (PCNA, ab92552; PLZF, ab189849, Abcam), meiosis (SYCP3, ab15093; STRA8, ab49602, Abcam; REC8, D222997; MLH1, D121003; DMC1, D224646, BBI Solutions), the blood‐testis barrier (β‐catenin, ab32572; ZO‐1, ab96587, Abcam), an apoptosis‐associated protein (Bax, ab32503) and sperm quality (PGK2, ab183031; HSPA4L, ab87241, Abcam).

ZO-1 and occludin in the colon were assessed by a Western blot. RIPA buffer (Thermal Scientific, USA) was used to homogenize the colon tissue. Then, the sample was centrifuged and prepared to measure the protein concentrations by BCA kits (P1511, Applygen Technologies Inc., Beijing, China). Each sample was loaded to the 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. After being blocked for 4 h in a Tris-buffered saline/Tween 20, the membrane was incubated with primary antibodies of ZO-1 (1:1000; ab96587, Abcam), occludin (1:1000; ab216327, Abcam), and β-actin (1:5000; ab8227, Abcam) at 4 °C overnight. After that, the membrane was incubated with the secondary antibody. The immunoreactive protein bands were visualized using a Clarity Western ECL substrate kit (Sigma, CA, USA). Scion Image (Frederick, MD, USA) was used to perform the densitometry analysis of protein bands. The values were normalized against the intensity of β-actin (Figure 1).

The localization of PARK7, ZO-1, and the cytoskeletal actin architecture was investigated by immunofluorescence staining on frozen biopsy samples and FHs74Int cells. After repeated washing with PBS, slides were permeabilized with Cytofix/Cytoperm (BD Pharmingen, San Diego, CA, USA) for 15 minutes at RT, washed with Perm/Wash Buffer solution (BD Pharmingen), and incubated with a primary antibody specific for PARK7 (ab18257; rabbit, 1 : 1000, Abcam, Cambridge, US), ZO-1 (ab96587; rabbit, 1 : 1000, Abcam), or Alexa Fluor® 546 phalloidin (7.5 units/mL, A22283; Thermo Fisher Scientific) for 1 hour at RT. In case of PARK7 and ZO-1 staining, slides were incubated with antirabbit Alexa Fluor 568®-conjugated secondary antibody (1 : 1000, A11036; Thermo Fisher Scientific) or antirabbit Alexa Fluor 488®-conjugated secondary antibody (1 : 1000, A21206; Thermo Fisher Scientific) for 30 minutes at RT. Thereafter, the slides were washed with a Perm/Wash Buffer solution and coverslipped with ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Sections were analyzed with an Olympus IX81 fluorescent microscope system.