Mouse anti pecam 1

Cells were fixed in PBS containing 4% formaldehyde, permeabilized in 0.1% Triton X-100, and blocked in 1% BSA containing 10% goat serum. Primary antibodies (incubated overnight) included rabbit anti- NF-κB p65 (Santa Cruz), mouse anti-phospho-tyrosine (4G10, Millipore), rabbit anti-Nck1/2 (Millipore), mouse anti-Nck (Abcam), goat anti-PECAM-1 (Santa Cruz), mouse anti-PECAM-1 (Cell Signaling Technology). Staining was visualized with Alexa-conjugated secondary antibodies (Invitrogen) and viewed on a Nikon Eclipse Ti inverted fluorescent microscope. Images were taken by using the Photometrics Coolsnap120 ES2 camera and analyzed by the NIS Elements BR 3.00, SP5 imaging software. Greater than 100 cells were counted for nuclear NF-κB for each condition of individual experiments. Proximity ligation assays were performed using manufacturer instructions, counterstained with DAPI and phalloidin, and quantified using NIS Elements software.

Sections of human samples were obtained from CCM lesions resected at University of Chicago using IRB approved protocols. Control samples were obtained from two autopsy subjects. Rabbit anti-KLF2 (1:200 Abcam ab203591), rabbit anti-KLF4 (1:50 Cell Signaling 4038S) and mouse anti-PECAM-1 (1:1000 Cell Signaling 3528S) were used for immunostaining.
Patient information pertaining to the human CCM samples studied is summarized below.
KRIT1 Familial: female, age 11, with the CCM1 Common Hispanic Mutation, removed due to CCM hemorrhage.
PDCD10 Familial: female, age 19, PDCD10 474+5G>A mutation, removed due to CCM hemorrhage.
Sporadic 1: female, age 24, solitary sporadic lesion, removed due to headaches and lesion growth.
Sporadic 2: male, age 31, solitary sporadic lesion, removed due to seizures.