Ary003b

For western blotting, homogenized tissues or whole-cell lysates,
samples were lysed in RIPA buffer containing protease inhibitor cocktail
(Roche) and phosphatase inhibitor cocktail (Roche). Cell lysates were
centrifuged at 14,000 x g for 15 min and supernatants were prepared in 4X
LDS Sample Buffer (Invitrogen) and separated by SDS-PAGE and transferred to
Immobilon 0.45μm membranes (Millipore). For Western blotting of
plasma samples, 1 μl of plasma was prepared in 2X sample buffer
(Invitrogen) with reducing agent, boiled, and analyzed using Western blot
against indicated protein. All primary antibody incubations were performed
overnight at 4 °C and secondary antibody incubations were performed
at room temperature for 2h. All primary antibodies were diluted at 1:1000 in
TTBS supplemented with 3 % BSA, except the ISM1 antibodies that were diluted
to 1:100. Secondary antibody incubations were performed at 1:10,000 dilution
in 1:1000 in TTBS supplemented with 5 % milk. Protein kinase array (R&D
systems ARY003B) was performed according to the manufacturers’
description. All antibodies used in this paper are described in STAR methods.

The profiling of phosphorylated proteins was determined using the proteome profiler human phospho-kinase array (R&D systems, ARY003B, Minneapolis, MN). Briefly, whole-cell extracts were incubated on the human phospho-kinase antibody arrays, and phosphorylation status was determined by subsequent incubation with anti-phosphotyrosine horseradish peroxidase as described by the manufacturer.

Protein kinase activity was analyzed using a human phospho-kinase array (R&D systems; catalog number ARY003B) to identify signaling pathways that were especially active in the KLA cells compared to LEC. Cell lysates (375μg protein) were prepared from confluent cells cultured in EGM-2MV media. Array assays were performed according to manufacturer’s protocol and imaged using Kodak X-ray film. Films were imaged on a scanner and signals quantitated by densitometry. Results from the array were confirmed by performing western blot analysis on cell lysates with antibodies for phospho-AKT (Cell Signaling Technology; Ser473 catalog number 4060 and Thr308 catalog number 2965), phospho-Proline-Rich AKT Substrate of 40 kDa (Cell Signaling Technology; p-PRAS40 Thr246; catalog number 22997), phospho-p44/42 MAPK (ERK1/2)(Cell Signaling Technology; catalog number 9101). Total AKT (Cell Signaling Technology; catalog number 9272), PRAS40 (Cell Signaling Technology; catalog number 2691), ERK-1/2 (Cell Signaling Technology; Cell Signaling Technology; catalog number 9102), and C4 actin (Chemicon; catalog number MAB1501) were also assessed.