Vegfr1

Cells were lysed in 0.5% Nonidet P-40 lysis buffer supplemented with protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Following electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane, Immobilon-P (Millipore Co., Milford, MA, USA). Membranes were blocked with Tris-buffered saline-Blotto/Blotto B (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hour and subsequently incubated overnight with antibodies directed against α-CaMKII, p-α-CaMKII, p-CREB, CREB, p-c-Jun, c- Fos, Lamin B1, HIF-1α, VEGFR-1, VEGFR-2 or β-actin (Santa Cruz Biotechnology and Cell Signaling Technology, Beverly, MA, USA). Signals were detected using a horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection kit (ECL; Amersham Biosciences, Pittsburgh, PA, USA) [14 (link)]. Band density was measured using ImageJ software and normalized to β-actin.

Tissues were fixed in 4% PFA, embedded in paraffin, and sectioned at 4–7 µm thickness. Immunostainings were performed using the following antibodies: α-SMA (1:500, Sigma-Aldrich, Cat. no A5228), CD31 (1:50, Dako, Cat. no M0823), VEGFR-1 (1:250, Santa Cruz Biotechnology, Cat no sc-31173), VEGFR-2 (1:250, Santa Cruz Biotechnology, Cat. no sc-6251) and VEGF-A (1:500, Santa Cruz Biotechnology, Cat no sc-7269). Photographs were taken with Olympus AX70 microscope (Olympus Optical, Tokyo, Japan). For whole-mount tissue imaging, 1 mm thick longitudinal sections were cut and stained with CD31 (1:100, Dako, Cat. no M0823) and α-SMA conjugated with Cy3 (1:500, Sigma-Aldrich, Cat. no C6198). For CD31, goat anti-mouse Alexa 488 (1:500; Invitrogen, Cat. no A-11001) was used. Imaging was performed by Nikon A1R multiphoton microscope (MPLSM) and LSM700 Zeiss confocal microscope (LSM).