CD164 modulates CXCL12-mediated migration by modulating CXCR4 downstream signaling pathways. p-Akt (A), p-PKC-ζ (B), and phospho-p44/42 MAPK (C) signaling in Jurkat cells nucleofected with control and CD164 siRNA duplexes. Cells were nucleofected, cultured for 48 hours, and stimulated with CXCL12 in the presence of fibronectin for 0, 1, 3, 5, and 10 minutes. (A) In control siRNA-treated cells, p-Akt signaling was induced and found to be maximal at 1 minute. This induction was significantly reduced in cells treated with CD164 siRNA. Total Akt and α-tubulin were used as loading controls with p-Akt protein levels compared with total Akt signal and were normalized to the Akt signal at 1 minute in cells treated with control siRNA (n = 2). (B) In control siRNA-treated cells, p-PKC-ζ signaling was induced at 1 minute and was maximal at 3 minutes after stimulation. This pattern was evident in cells treated with CD164 siRNA, but the activation of phosphorylated PKC-ζ was significantly attenuated. For example, after 3-minute CXCL12 stimulation, p-PKC-ζ levels were reduced to 28.5% ± 1.7% of the control levels. Total PKC-ζ (t-PKC-ζ) and α-tubulin were used as loading controls. Densitometry analysis of p-PKC-ζ protein levels compared with t-PKC-ζ in RNAi-treated Jurkat cells was normalized to the strongest p-PKC-ζ signal (3 minutes in control siRNA-treated cells; n = 3). (C) In control siRNA-treated cells, sp-p44/42 MAPK signaling was induced and found to be maximal at 5 minutes. The pattern and level of signaling induction were similar in CD164 siRNA–treated cells, indicating that reducing the level of CD164 did not significantly affect p-p-44/42 MAPK signaling. p44/42 MAPK and α-tubulin were used as loading controls with p-p44/42 MAPK protein levels compared with total p44/42 MAPK signal and were normalized to p44/42 MAPK protein levels at 5 minutes in cells treated with control siRNA (n = 2).