Ab181551

Total protein was extracted from testicular tissue. Western blot analysis was conducted as previously published by our laboratory [22 (link)]. The membrane was incubated with Anti-Integrin α6 (1 : 2000 dilution; ab181551, Abcam), Anti-Integrin β1 (1 : 2000 dilution; ab179471, Abcam), phospho-Akt polyclonal rabbit antibody at a dilution of 1 : 2000 (ab81283, Abcam), and phosphoinositide 3-kinase (PI3K) at a dilution of 1 : 2000 (4257, CST) overnight at 4°C and incubated with anti-rabbit IgG horseradish peroxidase conjugated secondary antibodies (1 : 3000 dilution; ab136817, Abcam). The relative protein expression was normalized with GAPDH (1 : 3000 dilution; ab8245, Abcam).

The protein used for WB is derived from the supernatant or cell lysate. Cell culture medium was concentrated by Amicon Ultra-4 Centrifugal Filter Devices (Amicon Ultra 50 K device, 50,000 MW CO, Millipore, USA) after centrifugation at 7500 × g for 40 min. The condensed samples (approximately 200 µl) were mixed with 5× loading buffer, boiled for 3 min and then analyzed by WB. Cell lysates obtained after treatment of cells with different inhibitors. AT2 cells were treated with PS1145 at 20 μM for 30 min, or JSH-23 (#S7351, Selleck Chemicals, China) at 6 μM for 1 h, or C646 at 20 μM for 1 h, or Anacardic Acid (#S7582, Selleck Chemicals, China) at 10 μM for 1 h, respectively. After 48 h, cell lysates were collected. The protein was separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to PVDF membranes (Millipore, USA), and blocked with 5% non-fat dry milk in TBST. After three times of washing with TBST, following primary antibodies dissolved in antibody buffer (Keygentec, China) were used: anti-ECM1 (sc-365335, SANTA CRUZ, USA), anti-p-p65 (S536) (#3033, CST, USA), anti-Ac-p65 (K310) (#3045, CST, USA), anti-t-p65 (#8242,CST, USA), anti-p300 (#86377, CST, USA), anti-integrinα6 (ab181551, Abcam, USA), anti-integrin β4 (ab236251, Abcam, USA), anti-ABCG1(ab218528, Abcam, USA), anti-HA (#3724, CST, USA), and anti-β-actin (#4970, CST, USA).

Approximately 1 × 10⁶ NALM6 cells were seeded on cover slips in 24-well plates and incubated for 24 h. Subsequently, the 80 μM S5 peptide was added into the culture medium and incubated with cells for 4 h, while the blank control group was added to the same volume of RPMI 1640 medium. After that, cells of each group were washed with PBS five times, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, Germany), and blocked with 1% BSA for 30 min. The cover slips were incubated with the anti-integrin α6 antibody (Abcam, ab181551) at 4°C overnight, followed by incubation with streptavidin-Cy3 (Thermo Fisher 434315, United States) and the goat anti-mouse Alexa Fluor 488 secondary antibody (Abcam, ab150113) for 1 h at room temperature in a dark chamber. Finally, cover slips were incubated with 1 μg/ml DAPI and mounted with slides with ProLong gold antifade (Invitrogen P26930, United States). Fluorescence images were visualized and captured under a confocal microscopy confocal laser-scanning system (Zeiss, LSM980, Germany) at 40× and 100× magnification. Co-localization was analyzed by ImageJ (http://rsbweb.nih.gov/ij/) and the co-localization finder plug-in.

At the end of imaging experiments, the mice were sacrificed by cervical dislocation and immediately dissected. The head part was collected, and haired skin and soft tissue were removed from the cranial surface. Paraffin sections (3 μm) were stained with hematoxylin and eosin (H&E) for routine histologic practice. Immunohistochemical (IHC) staining was performed following the conventional procedure, as we reported previously (Feng GK et al., 2019 (link)). Briefly, paraffin sections were dewaxed into xylene, rehydrated through graded alcohol, and microwaved for antigen retrieval. Blocking to inhibit the endogenous peroxidase activity and nonspecific binding, the sections were incubated with an anti-integrin α6 antibody (Abcam, ab181551, and 1:150) overnight at 4°C, followed by an HRP-conjugated polyclonal secondary antibody (1:200) at room temperature for 1 h. Finally, the positive immunoreactivity was visualized by staining with DAB (Zhongshan Jinqiao, ZLI-9017, China) and observed under a microscope (Nikon Eclipse, Japan).

Salicylic acid (Sigma-Aldrich, Dorset, UK) and Y-27632 (ROCKi; Enzo Life Sciences, Exeter, UK) were dissolved in growth medium or double distilled water, filter sterilized and used at the stated dose. SB203580 (Tocris Chemical Co., Bristol, UK) and KU55933 (R&D systems, Abingdon, UK), NS398 (Santa Cruz Biotechnology, Dallas, TX, USA) and celecoxib (Sigma-Aldrich, Dorset, UK) were dissolved in DMSO and used at the stated doses. The final concentration of DMSO was 0.1% vol/vol and DMSO alone was used as the vehicle control. PGE1 and PGE2 were purchased from Cayman Chemical (Michigan, USA). PGE1 and PGE2 were dissolved in 100% ethanol (Sigma-Aldrich, Dorset, UK) as a stock solution of 50 mg/mL and 100 mg/mL respectively and further diluted in 100% ethanol to the desired concentration. 0.1% Ethanol was used as the vehicle control. The function of Y-27632 was confirmed by Western blotting of the following antigens: NOTCH1 (ab87982) and Integrin α6 (ab181551) (Abcam, Cambridge, UK).

Paraffin-embedded skin samples from patients affected by bullous pemphigoid (N = 3), dermatitis herpetiformis (N = 3), and inflammatory EBA (N = 3) were sectioned (5 μm) for immunohistochemical analysis of GzmB (Cat # ab4059, ABCAM) and collagen VII (Cat # ab93350, ABCAM, Toronto, ON) using 3,3′-Diaminobenzidine for visualization, as well as α6 (Cat # ab181551, ABCAM) and β4 (Cat # ab182120, ABCAM) integrins, and collagen XVII (NC16a-3^{70 (link)}) visualized through Novared®. Healthy skin obtained from patients undergoing elective abdominoplasty was used as a control. In order to observe cellular infiltrate and tissue architecture, H&E staining was also performed using established methods. Following image acquisition, these H&E stained sections were de-stained by serial incubation in xylene (2 min), 100% EtOH (2 min), 95% EtOH (2 min), water (10 min), and acidic alcohol solution (10 min). In order to detect GzmB-producing cells, de-stained H&E sections were probed for GzmB (Cat # ab4059, ABCAM). All slides were scanned using a Aperio CS2 slide scanner (Leica, Concord, ON).