2.7. Western blotting

Expression levels of proteins involved in apoptosis were analyzed by Western blotting as previously described by our group.[26] In brief, SKBR3 and ZR75 cells were seeded in 100 mm petri dishes (Thermo Fisher Scientific, USA) and left to adhere overnight. Cells were treated with PAMAM dendrimers as well as lapatinib for 48 h. Cell lysates were collected and quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. NuPAGE® Bis-Tris Electrophoresis System (Thermo Fisher Scientific, USA) was used to run Western blotting. Briefly, stained protein samples were boiled at 95 °C for 10 min and equal amounts of protein (50 μg) were resolved in NuPAGE® Novex® Bis-Tris Gels (4–12%) (Thermo Fisher Scientific, USA) and electroblotted onto PVDF membranes, followed by blocking with 3% BSA (Thermo Fisher Scientific, USA). Then PVDF membranes were probed with the following primary antibodies; mouse anti-Bcl-2 (Abcam: abID# ab692), rabbit anti-Caspase-3 (Abcam: abID# ab13847), mouse anti-ErbB2 (Abcam: abID# ab16901), rabbit anti-phosphorylated ErbB2 for endogenous levels of ErbB2 at Tyrosine 877 (Abcam: abID# ab47262), rabbit anti-EGFR (Abcam: abID# ab131498), rabbit anti-phosphorylated EGFR on Tyrosine 1068 (Abcam: abID# ab40815), rabbit anti-ERK1/ERK2 antibody (Abcam: abID# ab115799), rabbit anti ERK1 (phospho Tyrosine 202) + ERK2 (phospho Tyrosine 185) (Abcam: abID# ab201015) and rabbit anti-JNK1/JNK2/JNK3 (Abcam: abID# ab179461). To ensure equal loading of protein samples, the membranes were re-probed with rabbit anti-GAPDH (Abcam: abID# ab9485). Following primary antibody staining, membranes were incubated with an anti-rabbit IgG-HRP (cat. no: 7074S, Cell Signaling Technology, Inc.) or anti-mouse IgG-HRP (cat. no: 7076S, Cell Signaling Technology, Inc.) secondary antibody. Immunoreactivity was detected using Pierce™ ECL Western Blotting Substrate (Peirce Biotechnology) by chemiluminescence. Blots were imaged using the iBright^TM CL1000 imaging system (Thermo Fisher Scientific, USA).

Relative quantification of protein expression was obtained by analyzing acquired Western blotting images using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The intensity of the bands relative to GAPDH bands was used to calculate a relative expression of proteins in each cell line.