2.4. Western blotting

Constitutive CCR4 degradation over the course of 6 h was assessed by incubating CHO‐CCR4 cells in serum‐free DMEM media containing 10 µg/ml cycloheximide at 37°C. The effects of the CCR4 ligands on CCR4 degradation were assessed by pre‐incubating CHO‐CCR4 cells for 30 min with 10 µg/ml cycloheximide prior to the addition of either CCL17 (100 nM) or CCL22 (100 nM) for 5 h at 37°C. Where inhibitors were used, cells were pre‐incubated with 10 µg/ml cycloheximide with the addition of either 43 or 1 µM Benzyl‐GalNac, DMSO vehicle control or 10 or 1 µg/mL tunicamycin for 30 min at 37°C. Cell lysates were generated and separated on 4–12% Bis‐Tris Protein gels as described previously.20 Gel transfer was performed using the iBlot dry transfer system. Non‐specific antigen binding was blocked using 5% milk powder in 1× TBST buffer for 1 h at room temperature. Blots were incubated overnight with 10E4 (diluted 1:2000 in blocking buffer) at 4°C. Following washing steps in 1× TBST cells were incubated with recombinant protein G‐HRP (diluted 1:4000 in 1× TBST) for 1 h at room temperature. Membranes were developed by chemiluminescence using ECL Plus substrate and imaged using a myECL imager. To obtain a loading control, membranes were stripped and re‐probed with GAPDH primary Ab overnight in blocking buffer.

In experiments comparing the constitutive degradation of WT CCR4 and CCR4‐Δ40 constructs it was observed that staining of the loading control GAPDH also degraded markedly over time in the presence of cycloheximide. In these experiments, the intensity of CCR4 staining was therefore normalized to the 0‐hour time‐point for each construct rather than to the GAPDH loading controls, which nonetheless demonstrated consistent protein loading for the first 3 h of the time‐course.