Imagequant tl 7

Each tissue lysate sample was electrophoresed through a precast gel (NuPAGE, 4–12% Bis-Tris Gel, 1.5 mm, Invitrogen, Carlsbad, CA). Proteins were transferred from the gel to a polyvinyldifluoride (PVDF) membrane (Millipore, Bedford, CA) and blocked with 5% milk in PBS (adjusted to pH 7.4) containing 0.05% Tween-20. The membranes were then separately incubated overnight with primary antibodies (1 ug/ mL) of anti- FSTL1, anti- Cathepsin B, anti- TGF-β, anti-PREX2 and anti-β-actin. After washing, the membrane was incubated with alkaline peroxidase-conjugated AffiniPure goat anti-rabbit IgG (111-035-003, Immuno Research) for 1 h (1:10,000). Proteins were detected with an enhanced chemiluminescent (ECL) system, and quantitative analysis of Western blotting was carried out using the ImageQuant-TL-7.0 software, version 2010 (Amersham Biosciences).

Confirmation of protein expression was performed by Western blotting. Each cell lysate sample (1 μg/μL, 10 μL) was electrophoresed through a precast gel (NuPAGE Novex 4–12% Bis-Tris Gel, 1.5 mm, 10 wells, Invitrogen, Carlsbad, CA). Proteins were transferred from the gel to a polyvinyldifluoride (PVDF) membrane (Millipore, Bedford, CA) by means of the semidry technique using the Criterion Blotter (Bio-Rad) at 100 V for 60 min and blocked with 5% milk in PBS (adjusted to pH 7.4) containing 0.05% Tween 20. The membrane was then incubated overnight with primary rabbit antibody (1 μg/mL) of anti-CD44 (1998-1, Epitomics, Inc.). After washing, the membrane was incubated with alkaline peroxidase-conjugated AffiniPure goat anti-rabbit IgG (111-035-003, Immuno Research) for 1 h (1 : 10000). Proteins were detected with an enhanced chemiluminescent (ECL) system, and quantitative analysis of Western blotting was carried out using the ImageQuant-TL-7.0 software, version 2010 (Amersham Biosciences).

The cell suspension was diluted with PBS (pH 7.2–7.4) until the cell concentration reached about 1 million/mL. Through repeated freezing and thawing or addition with tissue protein extraction reagents, the cells were destroyed to release the intracellular components. After centrifugation (3000 rpm, 15 min), the supernatant was collected. Western blotting was performed according to the method of Song et al. [28 (link)]. Briefly, samples were loaded onto a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) system (Beyotime) and transferred to polyvinylidene fluoride (PVDF) membranes electrophoretically (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline and Tween 20 for 1 h at room temperature. The membranes were then rinsed three times consecutively with a TBST buffer, and then incubated for 1 h with 1:1000 of primary monoclonal antibodies: β-actin, Nrf-2, HO-1, GCLC, p38, keap-1, JNK1/2 and ERK1/2 (Cell Signaling Technology, Shanghai, China) in a TBST buffer containing 1% skim milk. After that, secondary antibodies reacted with the bolts at room temperature for 2 h before visualization and analysis by densitometric scanning (Image Quant TL7.0, GE Healthcare Bio-Sciences AB, Mississauga, ON, Canada). Band density was quantified using ImageJ (National Institute of Health, Bethesda, MD, USA).

Caspase-2 variants were detected on one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to Stargardt et al. [44 (link)]. A ready-to-use molecular weight marker (Mark12™, Unstained Standard, Invitrogen, Waltham, MA, USA) was directly loaded as the size marker. The concentration of the soluble and insoluble fraction was calculated from densitometry analysis of SDS-PAGE gels using the software ImageQuantTL (7.0) (Cytiva, Uppsala, Sweden) via linear regression analysis. Purified T7AC-cpCasp2 (75, 50 and 25 µg/mL) was used as the standard. Two replicates of each sample were analyzed two times and the maximal coefficient of variation of 10% was used as acceptance criterion.