Megalin

Isolation and culture of primary proximal tubule cells was performed using collagenase digestion and sieving of renal cortex as previously reported with modifications [41 (link)–43 (link)]. Tubules were collected and cultured in hormonally defined serum-free DMEM/F12 (Gibco) medium supplemented with epidermal growth factor (EGF, 10 ng/ml), triiodothyronine (T3, 5 × 10^–12 M), selenium (50 nM), transferrin (5 μg/ml), insulin (5 μg/ml), hydrocortisone (50 nM) and streptomycin (100μg/ml) /penicillin (100 IU/ml, Gibco). Identification of proximal tubular cells was confirmed by immunostaining of a proximal tubule marker, megalin (1:200, Santa Cruz). Cells were then treated with PF and/or TGF-β1 as described above.

Tissue sections were incubated at room temperature for 90 minutes with a blocking solution containing 1% bovine serum albumin (BSA) fraction-V (Sigma, St. Louis, MO) and 0.5% Triton X-100 (Sigma) in PBS. At the end of the incubation, the blocking solution was removed and a mixture of the primary polyclonal antibodies was applied. megalin (H-245) (1:500, Cat. # sc-25470, affinity purified rabbit polyclonal antibody, raised against amino acids 4411– 4655 of megalin of human origin, Santa Cruz Biotechnology, Santa Cruz, CA), and cubilin (Y-20) (1:500, Cat. # sc-20607, affinity purified goat polyclonal antibody raised against a peptide mapping near the C-terminus of cubilin of human origin, Santa Cruz Biotechnology, Santa Cruz, CA) were applied for 48 hours at 4°C.
Following a 3 ×15 minutes PBS washing step, sections were incubated in a mixture of secondary antibodies (1: 1000): donkey anti-rabbit antibody labelled with Alexa 488, and donkey anti-goat antibody labeled with Alexa 594 (Life Sciences) for 2 hours. Then the tissue sections were washed with PBS (3 × 15 minutes) and covered with aqua soluble mounting media (Prolong diamond antifade mountant, Invitrogen) containing DAPI to visualize cell nuclei.