Bca protein assay kit

Quantitative measurement of MMP-9, D1R, and Cav1.2 in such rat brain structures as the vSTR was performed using a Rat MMP-9 ELISA Kit (Reddot Biotech, Kelowna, BC, Canada), a Rat Dopamine Receptor D1 (DRD1) ELISA kit (Reddot Biotech, Kelowna, BC, Canada), and Rat Voltage-dependent L-type Calcium Channel Subunit Alpha-1C ELISA Kit (Bioassay Technology Laboratory, Shanghai, China), respectively, following manufacturers’ protocols. Firstly, frozen brain structures were homogenized in cold buffer at pH 7.4 (0.32 M sucrose, 1 mM HEPES, 1 mM MgCl₂, 1 mM NaHCO₃, and 0.1 mM PMSF) containing cocktails of protease and phosphatase inhibitors (Sigma-Aldrich, Saint Louis, MO, USA), using a homogenizer ball (Bioprep-24, Allsheng, China) (10 s at 10,000 rpm). Then, homogenates were centrifuged for 5 min at 5000× g, the supernates were immediately removed, and protein concentration in the supernates was measured using a bicinchoninic acid assay (BCA) protein assay kit (Serva, Heidelberg, Germany). From each sample, 100 µg of protein was used in each ELISA assay. All data are expressed in ng/mL.

2D cultures and 3D constructs were lysed with RIPA buffer (R0278, Merck) containing protease inhibitor cocktail (11836153001, Roche, Basel, Switzerland). Total protein content was quantified with a BCA protein assay kit (39228, Serva, Heidelberg, Germany) and 5 or 10 μg of protein were loaded into 8% polyacrylamide gels and run by applying 225 V for 40 min. Afterwards, proteins were transferred to a PVDF membrane (IPVH07850, Merck) by applying 40 V for 2 h. The membrane was then blocked for 1 h with 4% (w/v) nonfat powdered milk in 0.2% PBS-Tween. Next, the membrane was incubated with primary antibodies anti-EGFR (700308, Invitrogen) at 1:1000 and anti-GAPDH (649201, Biolegend, San Diego, CA, USA) at 1:2000 for 1 h at room temperature. The membrane was then washed and incubated with secondary antibodies anti rabbit-HRP (ab6721, abcam) and anti mouse-HRP (ab6820, abcam), both at 1:1000 for 1 h at RT. Finally, the membrane was revealed for HRP detection with a SuperSignal West Pico Chemiluminescent Substrate (34080, Thermo Fisher Scientific, Waltham, MA, USA). Chemiluminescent images were taken in the ImageQuant^TM LAS 4000 mini (GE HealthCare, Chicago, IL, USA). Protein bands were quantified using ImageJ software and expressed as a ratio between the protein of interest and the loading control. Each blot was repeated three times (N = 3).

Quantitative measurement of MMP-9 in tissue homogenates was performed using a Rat Matrix Metalloproteinase 9 (MMP-9) ELISA Kit (Reddot Biotech, Kelowna, BC, Canada), following manufacturer’s protocol. Firstly, homogenates (see Section 2.5.1. Western blot) were centrifuged for 5 min at 5000× g. After this, the supernates were immediately removed and protein concentration was determined in each sample with a bicinchoninic acid (BCA) protein assay kit (Serva, Heidelberg, Germany). From each sample, 100 μg of protein was used in the ELISA assay. All data were expressed in ng/mL.