Cx3cr1

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).

BMM were treated for 24 h with 100 ng/ml LPS, after which the cells were washed twice with PBS and lysed in ice-cold Nonidet P-40 (NP-40) lysis buffer (50 mM Tris–HCl pH7.4, 100 mM NaCl, 10 mM NaF, 1 mM Na₃PO₄, 10 % glycerol, 1 % NP-40). After a 10 min incubation on ice the lysates were collected and boiled in sample buffer containing DTT. Samples were thereafter analyzed by SDS-PAGE. All proteins of interest were separated on 12 % polyacrylamide SDS gels. Proteins were transferred to 0.2 μm nitrocellulose membranes using the Trans-blot Turbo transfer system (Biorad). Nitrocellulose membranes were subsequently blocked in 5 % (w/v) non-fat milk in Tris-buffered saline (TBS) and incubated with CX3CR1 (Santa Cruz) and anti-α-tubulin (Cedarlane) primary antibodies overnight at 4 °C, followed by horse radish peroxidase-labelled secondary antibodies (Bio-Rad) for 1 h. Proteins were visualized with an enhanced chemiluminescence (ECL) detection system (Thermoscientific) and quantification of signal was performed using intensity measurements in ImageJ software.

Antibodies to mouse podoplanin were homemade (Breiteneder-Geleff et al., 1999 (link)); antibodies to CD31 were purchased from R&D Systems; and antibodies to CD9, CD63, CX3CR1, and CX3CL1 were from Santa Cruz Biotechnology, Inc. or Abcam. Immunofluorescence staining was performed on 4-µm paraffin sections of kidneys from renal transplant rejection (n = 6), synovias of rheumatoid arthritis (n = 4), intestines of Crohn’s disease (n = 4), skins of atopic dermatitis (n = 3), and skins of oxazolone-induced hypersensitivity dermatitis (n = 3) as described previously (Kerjaschki et al., 2004 (link)).
Secondary antibodies (Alexa Fluor 488, Alexa Fluor 594, or Alexa Fluor 633) were purchased from Molecular Probes. Images were acquired on an inverted confocal LSM700 (Zeiss) microscope with a 20× 0.8 NA air Plan Apochromat objective (ZEISS) for perivascular localizations of proteins or a 63× 1.4 NA oil Plan Apochromat objective (ZEISS) for intracellular localizations of proteins. Zen 2012 software (ZEISS) was used for image acquisitions. The perivascular distribution of CD9-associated immunofluorescence was plotted with the Fiji imaging software (Schindelin et al., 2012 (link)), and the relative fluorescence diameters were calculated.