Ab129067

Staining was performed on 2-μm-thick paraffin kidney sections that were pretreated with a heat-based antigen retrieval approach. The primary antibodies used were Col3a (Abcam, #ab-6310), LOX (Abcam, #ab-60178), α-SMA (a specific marker of myofibroblasts) (Abcam, #ab-7817), Pan Cytokeratin (a specific marker of epithelial cell) (Abcam, #ab-7753) and CTR1 (Abcam, #ab-129067). The sections were incubated with the primary antibodies at 4 °C for 1–2 nights, followed by incubation with biotin-labeled secondary antibodies and horseradish peroxidase (HRP)-labeled tertiary antibodies (DAKO). The sections were developed using a 3,3′-diaminobenzidine (DAB) kit (Vector, #SK4100), counterstained with hematoxylin, dehydrated, and examined under a light microscope. For colocalization immunofluorescence staining, the slides were incubated with Alexa Fluor conjugated secondary antibody (Invitrogen, #Alexa Fluor 568-A11011, Alexa Fluor 488- A11029) for 2 h at room temperature. Zeiss confocal microscope was used to observe the labeled slides.

Protein electrophoresis of whole cell lysates and transfer onto PVDF membrane was performed as previously described [45 (link)]. Membranes were incubated with 10% skim milk powder in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, prior to washing once with TBST and incubating with antibodies against CTR1 [46 (link)] (rabbit anti-SLC31A1, ab129067, Abcam, Melbourne, VIC, Australia), PARP (rabbit anti-PARP antibody 9542, Cell Signaling Technology, Danvers MA, USA), or GAPDH (mouse anti-GAPDH, Abcam, Cambridge, UK). Membranes were washed and incubated with anti-rabbit or anti-mouse dilution of horseradish peroxidase-conjugated anti-antibody for 60 min. Blots were developed with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, Rydalmere, Australia) and analysed using QuanityOne (Version 4.6.9, Bio-Rad, Gladesville, Australia). Both CTR1 and PARP protein expression were normalized to that of the housekeeping protein GAPDH.

Total protein was extracted by cell lysis buffer supplemented with protease inhibitor. Protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in TBS-T, membranes were incubated with the primary antibody (#ab129067, CTR1, Abcam, San Francisco, CA, USA). Protein was detected with Image Acquisition using ImageQuant LAS 4000 (Pittsburg, PA, USA).
For immunofluorescence assays, cells were cultured on glass slides with cell abundance of 30% in 6-well polystyrene microplates. The cells on glass slides were fixed with 4% formaldehyde and then permeabilized with PBS containing 0.2% Triton X-100 after washing three times in PBST. After blocking with 1% BSA in PBS, the cells were incubated with primary antibody (#sc-66847, CTR1, Santa Cruz, CA, USA) overnight at 4 °C. Cells were washed three times in PBS, followed by incubation with 2 μg/mL Alexa Fluor 594 phalloidin (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Cells were washed with PBS three times, nuclear stained with 5 μg/mL DAPI, and analyzed with a confocal laser scanning microscope (Olympus FluoView FV1000 confocal microscope, Melville, NY, USA).