Gtx627408

Western blotting was performed as previous reported^{70 (link)}. Briefly, equal molarity of protein extracts was loaded and separated in an SDS–PAGE, and transferred to a PVDF membrane. Immunoblot analysis was performed with overnight incubation of anti-vimentin (1:1000; 5741, Cell Signaling), anti-claudin1 (1:1000; 4933, Cell Signaling), anti-Snail (1:1000; 3879, Cell Signaling), anti-Twist1 (1:1000; 711565, Invitrogen), anti-ZEB1 (1:1000; 3396, Cell Signaling), anti-ZEB2 (1:1000; 97885, Cell Signaling), anti-ATP1A1 (1:10000; 14418-1-AP, Proteintech), anti-ATP1A2 (1:1000; 16836-1-AP, Proteintech), anti-ATP1A3 (1:1000; 28030-1-AP, Proteintech), anti-EMC1 (1:1000; GTX119884, Genetex), anti-ITGB1 (1:1000; 12594-1-AP, Proteintech), anti-αSMA (1:1000; ab7817, abcam), anti-collagen1 (1:1000; ab34710, abcam), anti-Flag (1:1000; F3165, Sigma), anti-p-p65 (S536) (1:1000; 3033, Cell Signaling), and anti-p65 (1:1000; 3034, Cell Signaling). anti-GAPDH (1:10000; GTX627408, Genetex) was used as loading controls for total cell lysates or cytosolic fraction. anti-flotillin1 (1:1000; ab133497, abcam) was used as loading control for membrane fraction.

Cells were collected by trypsinization and washed 3 times with PBS (1,000 × g, 5 minutes, 4°C). Cell pellets were resuspended in lysis buffer (1% (w/v) digitonin, 1× Roche cOmplete protease inhibitor, 0.5 mM PMSF, 10 mM Tris–HCL (pH 7.4)) and incubated on ice for 40 minutes. The lysates were then centrifuged (17,000 × g, 10 minutes, 4°C) and the post-nuclear fractions were transferred to new tubes. The protein concentration of each sample was determined by Bradford assay. Samples were adjusted with TBS buffer and 6× Laemmli buffer + 100 mM dithiothreitol (DTT) and heated at 50°C for 10 minutes. Samples were separated by SDS-PAGE and transferred to PVDF membranes (Merck), then blocked in 5% milk + PBS-T (PBS + 0.2% (v/v) Tween-20) for 1 hour. Blocked membranes were incubated with primary antibody (LRRC15 [LSBio aa393-422], ACE2 [Abcam Ab108252], GAPDH [GeneTex GTX627408]) in 5% milk + PBST (PBS + 0.2% (v/v) Tween-20) at 4°C overnight, then incubated with peroxidase (HRP)-conjugated secondary antibodies (Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) [Jackson ImmunoResearch 111-035-144], Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) [Jackson ImmunoResearch 115-035-146]) for 90 minutes at room temperature.

In all, 1.6 × 10⁶ of SH-SY5Y cells were treated with ADP-1 or ADP-3 (3 μM) for 24 h and then exposed to UV light (mercury lamp with 345−385 nm bandpass filter; average power: 8.24 mW/cm²; duration: 3 min). After washing with culture medium (Dulbecco’s Modified Eagle Medium/F12 supplemented with 10% FBS), cells were further incubated and collected the cell lysates in RIPA buffer at different time points. Cells lysates were first sonicated by UP200S (Hielscher Ultrasonics, Germany) and the total protein concentration in the resultant lysates was measured by detergent compatible protein assay (Bio-Rad, USA). In all, 50 μg of proteins were loaded onto nitrocellulose membrane (0.1 μm, GE healthcare, USA) adopted with the PR648 Slot Blot Blotting Manifold. Collected membranes were blocked by 2% bovine serum albumin and then stained with A11 antibody (AHB0052, ThermoFisher) and GAPDH antibody (GTX627408, GeneTex), respectively. To quantitate the A11 kinetics, the collected A11 signals were first normalized to internal control GAPDH and the resultant signals were then compared to the signal at 0.5 h-incubation to learn the relative fold value.

Cellular protein lysates were prepared by homogenization with 1× RIPA lysis buffer (Cell Signaling Technology, Billerica, MA, USA). Protein concentrations were measured using a protein assay dye (Bio-Rad Laboratories, Hercules, CA, USA). SDS-PAGE and immunoblotting analysis were performed as described previously [40 (link)]. The detecting antibodies were raised against E-cadherin (ab76055, Abcam, UK), vimentin (2707-1 epitomics), α-SMA (ab5694, Abcam, Cambridge, UK), fibronectin (ab2413, abcam), claudin 5 (ab15106, Abcam, Cambridge, UK), and GAPDH (GTX627408, GeneTex, Irvine, CA USA).