Imagequant las 500 system

Differentiated 3T3-L1 cells were lysed in PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Seong-nam, Korea) supplemented with a phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). Following quantification, 40 μg of protein sample was loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies for 2 h, followed by horseradish peroxidase-conjugated secondary antibodies (Promega, Madison, WI, USA) for 1 h. Protein bands were visualized using the ImageQuant ^TM LAS 500 system (GE Healthcare, Upsala, Sweden).

Whole cell protein lysate was extracted using Radio-Immunoprecipitation Assay (RIPA) buffer (Sigma Aldrich, St. Louis, MO, USA) plus protease and phosphatase inhibitor cocktail (Sigma-Aldrich). The SDS-PAGE consisting of 12% Acrylamide/Bis gel was used with equal amounts of protein samples. Membranes were incubated for one overnight with the primary antibodies against SOX2 (α-rabbit; 1:1000; Cell Signaling—D6D9, Danvers, MA, USA) or SOX9 (α-rabbit; 1:1000; Cell Signaling—D8G8H). The secondary antibody horseradish radish peroxidase (HRP; Cell Signaling—7074S) was incubated for 1 h at room temperature. Subsequently, signal was then detected by ImageQuant LAS500 system (GE Healthcare Life Science, Chicago, IL, USA) using LumiGLO^® chemiluminescence solution (Cell Signaling—7003S). Beta-Actin (alpha-rabbit; 1:1000; Cell Signaling—D6A8), and/or GAPDH (α-rabbit; 1:1000; Cell Signaling—D16H11), were used as loading for both quantity and quality control of protein lysates.

The cell extract was subjected to SDS-PAGE under reducing conditions, and the separated proteins were transferred to polyvinylidene fluoride transfer membranes. The membranes were incubated with an anti-phospho-p44/42 MAPK antibody or anti-phospho-AKT antibody (Cell Signaling Technology Inc., Beverly, MA, USA) at 4°C overnight. The membranes were washed and incubated with HRP-conjugated anti-rabbit IgG antibody or HRP-conjugated anti-mouse IgG antibody (American Qualex, San Clemente, CA, USA). After washing, the blots were visualized by enhanced chemiluminescence and detected using an ImageQuant LAS 500 system (GE Healthcare). The same membranes were re-probed with the anti-β-actin antibody (Sigma Chemical Corp., St. Louis, MO, USA), anti-p44/42 MAPK (Erk1/2) antibody, or anti-AKT antibody (Cell Signaling Technology) to confirm equal loading of the proteins. All western blot analyses were performed in triplicate.

Western blot analysis was performed with whole cell lysates as described previously [8 (link)]. Membranes were incubated in enhanced chemiluminiscence solution (Thermo Scientific, Germany) and developed with the ImageQuant LAS500 system (GE Healthcare, Germany). Immunofluorescence staining was done as described elsewhere [7 (link)], and pictures were taken with the Fluorescence Microscope BX50F, Olympus XC30 Camera and cellSens Entry imaging software (Olympus, Germany). Antibodies and dilutions that were used for Western blot analysis and immunofluorescence staining are listed in Additional file 2.

Protein was isolated from whole tissue lysate of thymus and spleen following standard protocol and estimated using Bradford reagent (Bio-Rad). Protein samples (100 µg) were mixed with loading buffer (5x), ran on 10% SDS-PAGE, transferred to PVDF membrane (Pall Corporation, USA) and probed overnight with Anti-ZFP67 and Anti-RUNX3 rabbit polyclonal antibody (Sigma), FoxP3 mouse monoclonal antibody (Merck-Millipore, USA) at 4 °C and developed with HRP-conjugated goat anti-rabbit and anti-mouse IgG (Santa Cruz Biotech, CA, USA) using chemi-luminescence substrate (Merck-Millipore, USA) under ImageQuant LAS 500 system (GE Healthcare, USA). The densitometric analysis was done using Image-J. Band intensities were normalized with GAPDH band intensity. Primary antibodies were used in 1:2000 and secondary antibodies were used in 1:3000 dilution.

For Western blot analysis, cells were lysed in RIPA buffer (20 mM Tris pH 8.0, 0.1% SDS, 150 mM NaCl, 0.08% sodium deoxycholate, and 1% NP40 supplemented with 1 tablet of protease inhibitor (Complete ultra mini-tablet, Roche, Indianapolis, IN, USA) and phosphatase inhibitor (PhosStop tablet, Roche, Indianapolis, IN, USA)). A total of 20 µg of total protein was loaded per lane, and protein was separated using SDS-PAGE. The separated proteins on the gel were transferred onto a PVDF membrane and were probed for specific antibodies against Rb (cat#9390; Cell signaling, Danvers, MA, USA), phospho-Rb (cat#8516, Cell signaling, Danvers, MA, USA), E2F2 (ab209662; Abcam, Cambridge, UK), HK1(cat#2024; Cell signaling, Danvers, MA, USA), and GAPDH (cat#5174; Cell signaling, Danvers, MA, USA) at 1:1000 dilution in 5% BSA in 1× TBST, overnight at 4 °C. After 4 washes with 1× TBST for 10 min, membranes were incubated with HRP-conjugated anti-mouse (cat#7076; Cell signaling, Danvers, MA, USA) or anti-rabbit antibodies (cat#7074; Cell signaling, Danvers, MA, USA) at 1:2000 dilution for 2 h. Images were visualized using the Image Quant LAS 500 system (GE Healthcare Life Sciences, Piscataway, NJ, USA).