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. Author manuscript; available in PMC: 2009 Aug 1.
Published in final edited form as: Cytokine. 2008 Jun 12;43(2):114–123. doi: 10.1016/j.cyto.2008.04.015

Fig. 6.

Fig. 6

Effect of lysosome and proteasome inhibitors on G-CSFR internalization in human neutrophils. Human neutrophils were isolated from whole blood using Ficoll-Paque PLUS, 3% Dextran sulfate, and ACK lysis buffer. After isolation, neutrophils were left untreated, or pre-treated with the following inhibitors for 1 h at 37ºC: lactacystin (100 μM) alone, chloroquine (10 μM) alone, or MG132 (5 μM) alone. Neutrophils were then stimulated with or without G-CSF (100 ng/ml) for 1 h at 4ºC, then transferred to 37ºC for the indicated time points. Cells were then stained using PE-conjugated anti-G-CSFR antibody for 45 min at 4ºC, fixed in 1% paraformaldehyde, and analyzed. Unstained controls are indicated in the solid grey shading. A shift to the left is indicative of decreased G-CSFR expression and consistent with G-CSFR internalization/degradation.