Rabbit anti pink1

The primary HSCs and human LX-2 cells were treated with RIPA buffer (Sigma-Aldrich) containing 1 mmol/L phenylmethanesulfonyl fluoride (PMSF: Sigma-Aldrich), vertexed, and centrifuged to collect the supernatant. The protein concentration in the supernatant was determined using the Bradford assay. The western blot analysis was performed as previously described [29 ]. The following primary antibodies were used in this study: rabbit anti-α-SMA (Cat. no. CY5295, 1:1,000, Abways), rabbit anti-collagen 1 (Cat. no. AF7001, 1:300, Affinity Biosciences, OH, USA), rabbit anti-PINK1 (Cat. no. DF7742, 1:500, Affinity), rabbit anti-parkin (Cat. no. sc-32282, 1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-P-parkin (Cat. no. AF3500, 1:500, Affinity), rabbit anti-TOM20 (Cat. no. ab186735, 1:1,000, Abcam, Cambridge, MA, USA), and GAPDH (Cat. no. AF7021, 1:5,000, Affinity). The immunoreactive bands were visualized using an Odyssey infrared imaging system (LI-COR Biosciences, Nebraska, USA). For protein quantification, the bands were scanned and quantified using Image-Pro Plus 6.0 software (Datacell, UK).

RIP assays were conducted using the Magna RIP Kit (Millipore, Billerica, MA, USA) [20 (link), 21 (link)]. HCT116 cells (2 × 10⁷/sample) were homogenized with RIP Lysis Buffer, followed by incubation with protein A/G magnetic beads coated with rabbit anti-PINK1 (Affinity) or rabbit anti-IgG (Millipore) overnight at 4 °C. The beads were washed with RIP Wash Buffer six times and incubated with proteinase K buffer to digest proteins. RNAs bound to PINK1 were extracted, purified, and analyzed by real-time PCR.