Icam 1

ß-Actin (RRID: AB_626632; cl C-4;1:500; Santa Cruz, Heidelberg, Germany).
Fra-2 (RRID: AB_2107084; cl Q-20;1:200; Santa Cruz, Heidelberg, Germany).
HCAM (RRID: AB 627,066; CD44; cl F-4; 1:200; Santa Cruz, Heidelberg, Germany).
ICAM-1 (RRID: AB_627123; cl G-5; 1:250; Santa Cruz, Heidelberg, Germany).
Integrin β4 (RRID: AB_626839; G-7; sc-13127; 1:500; Santa Cruz, Heidelberg, Germany).
L1-CAM (cl UJ 127.11; 1:25; obtained from Altevogt Lab, Heidelberg, (Ebeling et al. 1996 (link))).
ALCAM (RRID: AB_868825; ab49496; 1:500; Abcam, Cambridge, United Kingdom).

Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase and rabbit polyclonal antibodies specific for fractalkine/CX3CL1, CX3CR1, ICAM-1, VCAM-1, p-PI3K p85α, PI3K p85α, p-Akt, Akt, p-IKKα/β, IKKα/β, p-IκBα, IκBα, p-p65, p65, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant human fractalkine/CX3CL1 was purchased from PeproTech (Rocky Hill, NJ, USA). The short hairpin RNA (shRNA) plasmid used for gene knockdown was purchased from the National RNAi Core Facility Platform (Taipei, Taiwan). All siRNAs used were ON-TARGETplus siRNAs and purchased from Dharmacon Research (Lafayette, CO, USA). All other chemicals were obtained from Sigma–Aldrich (St. Louis, MO, USA).

Serial aortic root cryosections were stained with c-kit (CST), sca-1 (Abcam), ICAM-1 (Santa Cruz), and VCAM-1 (Santa Cruz). Images were captured using a fluorescent microscope (× 40).

Western blot was performed using testicular tissue as described in our previous study [33 (link)]. The primary antibodies included anti-activating transcription factor 4 (ATF4, Cell Signaling Technology, Danvers, MA, USA, 1 : 1000), anti-Bcl-2-associated X protein (Bax, Cell Signaling Technology, 1 : 1000), anti-B-cell lymphoma 2 (Bcl-2, Santa Cruz Biotechnology, 1 : 2000), anti-binding immunoglobulin protein (BIP, Cell Signaling Technology, 1 : 1000), anti-caspase12 (Cell Signaling Technology, 1 : 1000), anti-cleaved caspase3 (c-caspase3, Cell Signaling Technology, 1 : 1000), anti-C/EBP homologous protein (CHOP, Cell Signaling Technology, 1 : 1000), anti-GAPDH (Santa Cruz Biotechnology, 1 : 2000), anti-histone H3 (Santa Cruz Biotechnology; 1 : 1000), anti-intercellular adhesion molecule 1 (ICAM-1, Santa Cruz Biotechnology, 1 : 500), anti-inducible nitric oxide synthase (iNOS, Cell signaling Technology, 1 : 1000), anti-NRF2 (Santa Cruz Biotechnology, 1 : 1000), anti-pro-caspase3 (Cell Signaling Technology, 1 : 1000), and anti-vascular cell adhesion molecule 1 (VCAM-1, Santa Cruz Biotechnology, 1 : 500).

Six eyeballs were enucleated, and retinal tissues were collected and placed into Eppendorf tubes with 200 μL of lysate. The remaining tissues- “eyecups” were also mounted in tissue culture plate (Corning Incorporated, Corning, NY, USA) and up to 5 μL of lysate was added into the eyecups to extract retinal pigment epithelial proteins. After 5 min, the lysates were collected. Subsequent steps were performed in accordance with a conventional protocol. Equal amounts of protein samples were used for SDS-PAGE electrophoresis and transmembrane incubation with GLUT-1 (Millipore Corporation. St. Charles, MI, USA), ICAM-1, TNF-α (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and albumin (Abcam plc, Cambridge, UK) primary antibodies. The following day, secondary antibody incubation was conducted at room temperature for 1 h after the membranes were washed three times. Finally, the relative densities of the blots were measured by UVP GelDoc-It Imager (UVP LLC, Upland, CA, USA).