Figure 1.
Epo stimulation induces raft formation and aggregation.
(A) Dot blot detection of GM-1 in UT7 cell lysates in non-raft (fractions 5, 6) and raft fractions (fraction 2) with corresponding densitometry value in controls, and after Epo or MBCD treatment. Representative blot of at least three independent experiments. (B) Western immunoblot of Lyn in raft (R) (fractions 1–2) and non-raft (NR) fractions (fractions 4–6). Treatment with Epo increased Lyn kinase incorporation into raft fractions, whereas raft disruption by cholesterol depletion with MβCD precluded Lyn incorporation. Representative western of at least three independent experiments. (C) Immunofluorescence of UT7 cells showing an increase in raft (red) accumulation after Epo exposure. (D) Immunofluorescence of UT7 cells before and after Epo stimulation showing increased raft aggregates (red) in the plasma membrane and corresponding quantitation. (E) Immunofluorescence of primary erythroid bursts showing an increase in cellular membrane raft fluorescence intensity (red). Immunoflorescence experiments were repeated at least 3 times, representative micrographs displayed.
Figure 2.
Epo-R co-localizes with lipid rafts.
(A) Confocal immunofluorescence of cells untreated or after Epo stimulation, lipid rafts:red, Epo-R:green, DAPI/Hoechst:blue. Right panel is a merged image showing lipid raft and Epo-R co-localization (yellow). UT7 cells are shown in rows 1 and 2, while human primary burst forming units are shown in rows 3 and 4, followed by a maturing, enucleated erythroid precursor in row 5. (B) Three dimensional rendering of UT7 cells either untreated (left) or after Epo treatment (right). Top two rows display isosurfacing of the rafts (red), Epo-R (green), and nucleus (Dapi, blue). Dapi was removed from the middle row to further visualize association of the receptor with rafts in the second row of panels. The bottom row displays volume rendering of the same cells to illustrate membrane colocalization (yellow). (C) Quantitation of colocalization in human primary erythroid cells. Values represent mean ± SE. Immunofluorescence experiments were repeated at least 3 times, representative micrographs provided.
Figure 3.
Epo stimulation recruits signal effectors into raft fractions.
(A) Raft fractions (R) were separated from non-raft fractions (NR) and immunoblotted for Epo-R to investigate receptor translocation into rafts after Epo stimulation. Corresponding quantitation represents the mean ± SE of two independent experiments using four different Epo-R antibodies. (B) Raft fractions were isolated after stimulation with Epo at the indicated time points and immunoblotted for Epo-R. Results show that EpoR is recruited into rafts within 1 minute of Epo stimulation reaching maximum loading at 10 minutes, followed by gradual redistribution thereafter. Accompanying graphic quantitation of the representative experiment. (C) UT7 cells were starved overnight then treated with Epo for 10 min. After fractionation, the non-raft (NR) fractions and raft (R) fractions were pooled and immunoblotted for the indicated proteins. (D) Activated forms of Jak2, STAT5, and MAPK were also increased in the raft fractions after Epo stimulation. All westerns were repeated at least in duplicate.
Figure 4.
Raft integrity is necessary for Epo-induced signaling.
(A) UT7 cells were starved for 2 h then pretreated with MBCD for 30 min and stimulated with 3 U/ml Epo for 10 min; lysates were immunoblotted with the indicated antibodies. (B) UT7/Epo cells were starved for 2 h then pretreated with MBCD for 30 min and stimulated with 3 U/ml Epo for 10 min. Lysates were immunoblotted with P-Akt. The findings show abrogation of Akt phosphorylation following MBCD pretreatment. (C) UT7 cells were pretreated with MBCD for 30 min, then stimulated with PMA for 30 min. (D) UT7 cells were starved for 2 h then pretreated with Nystatin for 30 min and stimulated with Epo for 10 min. Immunoblots for phospho-STAT5, STAT5, and β-actin antibodies with densitometry analysis. All westerns are representative of at least 2 independent experiments.
Figure 5.
Cholesterol depletion attenuates Epo-induced STAT5 phosphorylation in primary erythroid progenitors.
(A) Bone marrow mononuclear cells from a normal donor were isolated then stained with CD71:APC, CD45:FITC, and P-STAT5:PE. CD71Hi/CD45dim cells representing erythroid progenitors were gated. (B) Graphic comparison of geometric mean florescence intensities, mean ± standard error from 3 independent experiments. (C) Representative flow histogram showing shift in phospho-STAT5 florescence intensity in primary erythroid progenitors treated with Epo with or without MβCD.
Figure 6.
Recruitment of Epo-R into lipid rafts is dependent on Rac1 and RhoA GTPase activation.
(A) Raft fractions were isolated from UT7 cells pretreated with 100 nM Rac1 inhibitor for 1 hr prior to Epo stimulation then immunoblotted for Epo-R with corresponding quantitation. (B) Raft fractions were isolated from UT7 cells pretreated with 100 uM ROCK inhibitor (Y-27632) for 1 h prior to Epo stimulation then immunoblotted for Epo-R with corresponding densitometry analysis. Westerns are representative of two independent experiments.