Image lab software version 3

According to the method of Kang et al. [13 (link)], homogenates of medaka gills or embryos were prepared. Aliquots containing 20 μg of homogenates were heated at 65°C for 25 min and fractionated by electrophoresis on SDS-containing 7.5% polyacrylamide gels. Transferred PVDF blots were incubated with VILL polyclonal antibody (1:200,000 dilution) or β-actin monoclonal antibody (1:10,000 dilution; 8226, Abcam, Cambridge, UK) for 2 hrs, followed by a 1-hr reaction with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (Pierce, Rockford, IL, USA). Blots were developed using the SuperSignal West Pico Detection Kit (#34082, Pierce) and analyzed with Image Lab software version 3.0 (Bio-Rad) after densitometric scanning of membranes using a ChemiDoc XRS+ (Bio-Rad). Results were converted to numerical values to compare relative intensities of immunoreactive bands.

EMSA assay was performed according to the modified method as described previously.⁴⁰ A double-stranded mutated oligonucleotide, which positive-sense strand was (5′-CTTAAAGTGCAGGAGCTCTGTGGATGTGCTGCT-3′), was used to evaluate the specificity of PLSCR1-binding site in the IP3R1 gene promoter region (5′-CTTAAAGTGCAGTAACCATGTGGATGTGCTGCT-3′), together with the complementary oligonucleotide (5′-AGCAGCACATCCACATGGTTACTGCACTTTAAG-3′) double-stranded probes. The anti-PLSCR1 antibody was used for supershift experiments. The results were photographed using a Bio-Rad phosphorimager and analyzed with Image Lab Software, Version 3.0 (Bio-Rad, Hercules, CA, USA).

The expressions of cytoskeletal proteins (ARP3, ARPC3), migration protein (MMP-9), and apoptotic protein (Caspase3) were detected by WB. Cal-27 cells were cultured with 0.60 mM ICD for 24 h, 48 h, and 72 h, respectively. All proteins were extracted with a RIPA buffer (Beyotime, Shanghai, China) containing a protease inhibitor (Beyotime, Shanghai, China) and a phosphatase inhibitor (Roche Diagnostics GmbH, Mannheim, Germany). The protein concentration of each group was measured using the BCA protein assay kit (BCA; Beyotime, Shanghai, China). After protein denaturation, equal amounts of proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were then enclosed in 5% skim milk powder at room temperature for 2 h and incubated with primary antibodies (ARPC3, ab37916, 1:1000; ARP3, ab181164, 1:1000; MMP-9, ab283575, 1:1000. Abcam; Caspase3, PA5-122288, 1:1000) at 4 °C overnight. The membranes were washed 3 times with 0.1% Tween-20 (TBST) and incubated with the appropriate HRP-chelated secondary antibodies (1:5000, Abcam, Cambridge, UK) at room temperature for 1 h. Bio-Rad Image Lab software (Version 3.0) was used to conduct an exposure analysis after ECL was added.

The total proteins in the CSF and serum samples were extracted with protein lysis solution (Tiangen Biotech). Protein concentration was measured with a bicinchoninic acid kit (Tiangen Biotech). SDS-PAGE sample buffer (Tiangen Biotech) was added to the protein samples, and the samples were boiled for 5 min. A total of 20 μg protein was used for SDS-PAGE with 10% acrylamide gels, and the protein was then transferred onto a membrane at a constant voltage of 100 V for 2 h under ice-cold conditions. The membrane was blocked with 5% skimmed milk for 1 h at room temperature. Rabbit anti-human monoclonal anti-VEGF primary antibody (1:1,000; cat. no. ab52917, Abcam, Cambridge, MA, USA) and the internal reference rabbit anti-human monoclonal anti-β-actin primary antibody (1:5,000; cat. no. ab115777, Abcam) were added for incubation with the membrane at 4°C overnight. A goat anti-rabbit polyclonal immunoglobulin G secondary antibody (1:3,000; cat. no. ab97047, Abcam) was subsequently added for incubation at room temperature for 1 h. The membrane was placed in an enhanced chemiluminescence solution (Tiangen Biotech) and then exposed to the Gel Doc™ EZ Imager (Bio-Rad Laboratories, Inc.). Image Lab software version 3.0 (Bio-Rad Laboratories, Inc.) was used for protein band analysis. The relative content of the target protein was determined as the ratio to the internal reference β-actin.

Western blot was performed as previously described (11 (link)). The primary antibodies used included anti-CD206 (ab64693, Abcam), anti-Arg-1 (ab233548, Abcam), anti-Ym1 (ab192029, Abcam), anti-SIRPα (ab191419, Abcam), anti-p-STAT3 S727 (ab32143, Abcam), anti-p-STAT3 Y705 (#9664, CST), anti-STAT3 (9662, CST), anti-α-SMA (ab7817, Abcam), anti-cleaved Caspase 3 (CST; 9662), anti-Caspase 3 (CST; #3498), anti-Bcl2 (CST; #3498), anti-E-cadherin (GTX100443, GTX), and anti-GAPDH (ab181602, Abcam). The secondary antibodies included Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) and Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (111035003, 115035003, Jackson, West Grove, PA, USA). Protein levels were quantified using the Image Lab software, version 3.0 (Bio-Rad, Hercules, CA, USA).