Versadoc 4000 mp

2-D protein extract analysis was carried out at the CICT following an adaptation of a previous report [40 (link)]. Protein extracts (125 µL) were loaded onto 7 cm immobilin strips, and the isoelectric focusing protocol was as follows: passive rehydration for 12 h at 20 °C (without pause); 50 V for 10 h (fast); 500 V for 1 h (fast); 1000 V for 1 h (fast); 4000 V for 30 min (linear); 400 V, 6000 V for 1 h (fast); and 500 V for 99 h (fast) (hold step), followed by separation in a 12% SDS–polyacrylamide gel as reported previously [40 (link)]. The gels were stained with Oriole (Bio-Rad, Hercules, CA, USA), and the image was captured with a Versadoc 4000 MP (Bio-Rad, Hercules, CA, USA). The gels were compared using the PDQuest^TM Analysis software (Bio-Rad, Hercules, CA, USA).

Gel images were taken with an imaging system (VersaDoc4000MP, Bio-Rad, USA) and analyzed by using PDQuest Advanced 2D-image analysis software (Bio-Rad, USA). The quantity of each spot was normalized using local regression model. Based on average spot volume ratio, spots whose relative expression levels were changed at least 2-fold (increase or decrease) among the compared groups were considered to be significant. Statistical significance was assessed by using student's t-test (P < 0.01). Protein spots that displayed statistically significant regulation were cut by using automated EXQuest Spot Cutter (Bio-Rad) and deposited in a 96-well plate for in gel-tryptic digestion.

Proteins of WSL and WSL-gRp30 cell lysates were separated by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, and blots were subsequently incubated as described^{48 (link)}. The anti-FLAG M2 monoclonal antibody (Sigma-Aldrich), and the α-tubulin specific monoclonal antibody B-5-1-2 (Sigma-Aldrich) were used at dilutions of 1: 5,000 or 1: 20,000, respectively. Binding of peroxidase-conjugated anti-mouse IgG antibodies (Jackson ImmunoResearch) was detected by chemiluminescence (Clarity Western ECL Substrate, Bio-Rad), and recorded (VersaDoc 4000 MP, Bio-Rad).

Samples from each group were collected in RIPA lysis buffer (50 mM Tris-Cl [pH 7.4], 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], and 150 mM NaCl), and a protease inhibitor tablet was added (Beyotime Sciences, USA). The samples were centrifuged at 12,000 rpm at 4°C, and protein concentration was determined using a BCA protein assay (CwBio Sciences, CN). Lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. The membrane was probed with β-actin horseradish peroxidase (HRP) (Proteintech, CN) and blocked with 5% BSA before incubation with the primary rabbit anti-mouse FGF21 antibody (Abcam, UK). An HRP-conjugated goat anti-rabbit IgG antibody (Millipore) was applied, and after incubation, the membrane was developed with an Amersham-enhanced chemiluminescence western blotting detection system (Bio-Rad, Versa Doc 4000MP, USA).

Total RNA was isolated using miRCURY™ RNA Isolation Kit (Exiqon¸Vedbaek, Denmark) according to the manufacturer’s protocol and as previously described [33 (link)]. Detection and quantity of RNA was determined using a Bioanalyzer and RNA 6000 Nano chips according to the manufacturer’s protocol (Agilent Technologies, Santa Clara, CA, USA).
For examination of the c-KIT gene expression, 500 ng of total RNA was converted into cDNA using RT² first Strand Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. For PCR amplification 1 μl of diluted cDNA (<500 ng) was used as a template to make 50 μl reactions containing the reagents from HotStarTaq Master Mix (Qiagen). KiCqStart™ primer pairs (Sigma-Aldrich) used were as follows: GAPDH (forward) 5'-CTTTTGCGTCGCCAG-3', (reverse) 5'-TTGATGGCAACAATATCCAC-3'; c-KIT (forward) 5'-ACAAAACCAGAAATCCTGAC-3', (reverse) 5'-CAGTTCCTGGACAAAAATACC-3'. The length of the expected amplicon for c-KIT and GAPDH were 109 and 139 base pair, respectively. PCR amplification involved 30 cycles at 94°C for 30 seconds, 55°C for 45 seconds, and 72°C for 30 seconds. PCR products were resolved on a 1.5% agarose gel (by loading 5 μl of the PCR product) and detected utilizing Gel Star® Nucleic Acid Gel Stain (Lonza, Rockland, ME, USA) and a VersaDoc 4000 MP (Bio-Rad Laboratories, Hercules, CA, USA).

After PDT treatment, UM-UC-3 and HT 1376 cells were washed twice with ice-cold PBS and harvested in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton X-100, 0.5% sodium deoxycholate (DOC), 0.1% SDS, 2 mM phenylmethanesulfonyl (PMSF), 2 mM iodoacetamide (IAD),) and 1× protease inhibitor cocktail (Roche, Indianapolis, IN, USA)). After centrifugation at 16,000 g for 10 min at 4°C, supernatants were used for protein quantification using the Pierce BCA Protein Assay Kit, followed by denaturation of the sample with Laemmli buffer. For the Western Blotting analysis, 60 µg proteins were loaded per lane on sodium dodecyl sulphate-polyacrilamide gels (SDS-PAGE). Following electrophoresis and transfer to PVDF membranes (Bio-Rad, Hercules, CA, USA), the blots were incubated in 5% (m/v) nonfat milk in TBS-T (20 mM Tris, 150 mM NaCl, Tween 0.2%, pH 7.6) and probed with rabbit anti-galectin-1 1∶1,000 (Abcam, Cambridge, UK), rabbit anti-GLUT1 1∶1,000 (Chemicon, Boston, MA, USA) and mouse anti β-actin 1∶20,000 (Sigma) antibodies. After washing, the membranes were probed with secondary anti-rabbit or anti-mouse IgG-HRP-linked antibodies (1∶10,000; Bio-Rad). Immunoreactive bands were detected by enhanced chemiluminiscence (ECL) substrate using an imaging system (VersaDoc 4000 MP, Bio-Rad) followed by densitometric analysis.