Qproteome mammalian protein prep kit

Cells treated with TMX, COH or combination treatment were harvested at the end of incubation period and lysed on ice for 30 min in RIPA buffer (Cell Signaling, #9806) containing a complete protease inhibitor cocktail (Roche, 11836145001) and PhosSTOP (Roche, 04906837001). The Qproteome Mammalian Protein Prep Kit (Qiagen) was used to extract protein from harvested tumors. The protein concentrations were determined using a Bio-Rad Protein Assay Kit (Bio-Rad, 500-0001). Approximately 40 μg of protein was mixed with an equal volume of 2 × SDS loading buffer, boiled for 5 min, then separated by Tris-glycine SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk in PBST (PBS containing 0.05% Tween 20) and incubated with primary antibodies at 4°C overnight. The membranes were then washed with three times with PBST for 10 min, and incubated with HRP (horseradish peroxidase)-labeled secondary antibodies for 2 h at RT. Immunoblots were visualized using the VersaDoc 5000 imaging system (Bio-Rad) and processed using quantity one (Bio-Rad).

Protein extraction from cultured cells was done using Qproteome™ Mammalian Protein Prep Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Cells were collected by centrifugation at 1500 rpm for 5 min at 4 °C, washed twice with ice-cold PBS, and then homogenized in lysis buffer supplemented with protease inhibitor and benzonase nuclease. Protein quantification was performed using a DC Protein Assay II kit (Bio-Rad, Germany).

Procollagen type 1 and MMP-13 were measured by ELISA and quantified in nanograms of protein. All protein samples were extracted from murine skin using the Qproteome Mammalian Protein Prep kit (Qiagen, Hilden, Germany), according to the manufacturer’s manual. The concentration of protein was determined by the Bradford assay [34 (link)]. The expression level of procollagen type 1 was determined with a mouse procollagen type 1 N-terminal propeptide (PINP) ELISA kit (MyBioSource, San Diego, CA, USA). The expression level of MMP-13 was determined with a mouse matrix metalloproteinase 13 (MMP-13) ELISA kit (MyBioSource). Samples and standards were incubated together with horseradish peroxidase (HRP)-conjugated reagent in pro-coated plates for 1 h. After the incubation, the wells were decanted and washed five times. The wells were then incubated with a substrate for the HRP enzyme. Finally, a stop solution was added to stop the reaction. The intensity of color was measured at 450 nm in a microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). The concentration of total protein was determined by comparing the optical density (OD) of the samples to the standard curve. The measurement was carried out in triplicate.

Petri dishes were used to culture and treat MDA-MB-231 cells (2 × 10⁵ cells/mL) with AELE (261 and 522 μg/mL) for 24 h. The Qproteome mammalian protein prep kit (Qiagen, Hildren, Germany) was used to extract the proteins, which were then quantified based on the Lowry method. Proteins were prepared for separation by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (10%) as explained by El Zein et al. [41 (link)], and transferred to Polyvinylidene fluoride membranes for protein assessment, as previously described [42 (link)]. Membranes were incubated with primary antibodies (1:1000) anti-cytochrome c (Abcam, Cambridge, UK), anti-p21 (Cell Signaling, Danvers, MA), anti-Bax and anti-Bcl2 (Elabscience, Houston, TX, USA). The internal loading control was detected using anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). After washing, the secondary antibody (Bio-Rad, Hercules, CA, USA) was added at the recommended concentrations (2:5000). Blots were visualized on ChemiDoc machine (BioRad, Hercules, CA, USA) and relative expression of proteins bands was quantified using the ImageJ program [43 (link)].

Western blotting analysis was performed using standard protocols as published elsewhere [10 (link), 14 (link)]. Briefly, protein lysates were extracted from the cells (1 × 10⁷ cells) using a Qproteome Mammalian Protein Prep Kit (Qiagen), and the lysates were applied to 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels for separation. Proteins were then transferred onto Immobilon-P membranes (Millipore, Billerica, MA, USA). The membranes were probed with primary and secondary antibodies using standard techniques. Anti-FPGS (cat. no. ab184564; Abcam, Cambridge, UK), anti-DHFR (cat. no. 872442; R&D Systems, Minneapolis, MN, USA), anti-caspase 3 (cat. no. 9665; Cell Signaling Technology, Massachusetts, USA), anti-PARP (cat. no. 9542; Cell Signaling Technology), anti-cleaved PARP (cat. no. 9541; Cell Signaling Technology), and anti-β-actin (cat. no. A2066l Sigma-Aldrich Japan) antibodies were used as primary antibodies, and anti-rabbit polyclonal antibodies (cat. no. 7074; Cell Signaling Technology, Tokyo, Japan) were used as secondary antibodies. Protein detection and quantification were performed using Amersham ECL Prime Western Blotting Detection Reagent and an ImageQuant LAS4000mini system (GE Healthcare Life Sciences, Little Chalfont, UK).