Figure 1.
Binding of Anti-EpoR antibodies to spleens from WT or human EpoR KI mice.
Spleens from untreated or rMsEpo-treated mice were sectioned and stained with anti-EpoR antibodies as described in Methods. Note the enlargement in spleen with rMsEpo addition due to enhanced erythropoiesis. (A) Staining with anti-EpoR monoclonal antibody A82. (B) The same sections as in A were stained with polyclonal antibody M-20. (C) Staining of mouse WT small intestine (ileum) with A82. Note the staining of the material in secretory granules and just under the surface. (D) A82 Western blot with mouse tissues. Note the ∼ 45 kDa protein non-EpoR protein in small intestine.
Figure 2.
Specificity to EpoR of anti-EpoR antibodies A82 and M-20.
EpoR protein expression analysis was performed by immunoblotting (IB) with anti-EpoR antibodies A82 or M-20 (lot E2004). Three identical blots using the same lysates were processed then probed with the indicated antibodies at the same time. The EpoR positive controls were a Cos-cell lysate overexpressing a FLAG-tagged version of Human EpoR [6] and UT-7/Epo cells. Negative controls were 769-P cells and COS cells transfected with an empty vector. The position of the 59 kDa full-length EpoR protein is indicated by the arrow. The positions of molecular weight markers are shown. The lower portion of the blot was also probed with anti-cyclophilin as a loading control.
Figure 3.
No EpoR detected by A82 or M-20 in normal and cancerous human breast tissues (Her2+ and Her2−) but M-20 detected a 59 kDa band in MCF-7 Cells.
EpoR protein expression analysis was performed by immunoblotting (IB) with A82 and M-20. Four identical immunoblots with the same master mix were performed at the same time and probed with different antibodies under the same conditions. Lysates included tissue samples from 3 different human tumor biopsies, a normal breast sample or MCF-7 cells. The positive control was UT-7/Epo cells and the negative control was 769-P cells. The positions of molecular weight markers are shown. (A) Anti-EpoR monoclonal antibody M-20 (lot E2004). (B) Anti-Her2 antibodies. (C) Anti-EpoR antibody A82. A blot with anti-GAPDH antibodies served as a loading control.
Figure 4.
The 59 kDa protein detected by M-20 In MCF-7 cells is not bound by other anti-EpoR antibodies.
The indicated lysates were immunoprecipitated (IP) then the immunoblotted (IB) with the indicated antibodies: ab10653 (abcam Inc), Mab307 (R&D systems), C-20 & M-20 (Santa Cruz Inc) or A-82 (Amgen Inc). COS cell lysates expressing a FLAG-tagged version of EpoR (FLAG-EpoR) [6] and UT-7/Epo cells served as EpoR positive controls. 769-P cells served as the EpoR negative control. (A) Westerns were immunoprecipitated (IP) with ab10653 or M-20 followed by immunoblotting (IB) with M-20. The position of full-length 59 kDa EpoR in positive controls is indicated by the arrow. Positions of molecular weight markers (kDa) are shown. Bands detected in 769-P lysates are non-EpoR cross-reacting proteins and include antibody chains that were not removed completely or protein G that leached from beads. Note the detection of a 59 kDa band with MCF-7 cells with the M:20/M:20 combination but not with the ab10653/IB:M-20 combination. (B) IP:IB combinations with the indicated antibodies were subjected to western analysis. The western slice containing the 59 kDa EpoR band from each combination is shown. Note the 59 kDa bands detected in EpoR positive controls but not 769-P cells. Only the M-20:M-20 combination detected a 59 kDa band in MCF-7 cells.
Figure 5.
Anti-EpoR westerns with A82 on normal and tumor cell lines and tumor tissues.
EpoR protein expression analysis was performed by immunoblotting (IB) with A82. Samples were processed and subjected to western using the anti-EpoR monoclonal antibody A82. With long exposures this antibody can specifically detect EpoR protein in cell lysates at levels as low as 100 fg (<20 molecules/cell) [23]. The positive control was erythroid progenitor cells. Positive control erythroid cells were prepared by culturing human CD34+ cells for 4–6 days in media that supported differentiation into Epo-responsive erythroid cells with physiologically relevant levels of EpoR5. The position of the 59 kDa full-length EpoR protein is indicated by the arrow. The proteins smaller than 59 kDa have been shown previously to contain EpoR sequences and are EpoR fragments [23]. Western with the indicated human normal and tumor tissue biopsies, primary cells and skin cell lines is shown. The blots were stripped and reprobed with anti-GAPDH antibodies to serve as a loading control. Positions of molecular weight markers are shown.
Figure 6.
Anti-EpoR westerns with A82 on normal and tumor tissues.
EpoR protein expression analysis was performed by immunoblotting (IB) with A82. The positive control was erythroid progenitor cells prepared as described in Figure 5. The position of the 59 kDa full-length EpoR protein is indicated by the arrow. Tissue biopsies from different human subjects with the indicated cancer types are shown. Samples were from cancerous regions (C), or adjacent regions containing normal cells (N) from the same patient. In this experiment skin tissue served as the negative control. The blots were stripped and reprobed with anti-GAPDH antibodies to serve as a loading control. Positions of molecular weight markers are shown.
Figure 7.
EpoR protein expression in breast and lung cancer cell lines.
Breast and lung cancer cell lines were grown and subjected to western blotting with A82. Positions of molecular weight markers are shown. Full-length EpoR is indicated by the arrow. 769-P and OCIM-1 served as negative and positive controls for EpoR respectively. Blots were stripped and reprobed with anti-cyclophilin B to control for protein loading. (A) Breast cancer cell lines. (B) Three of the lung cancer lines that had detectable levels of EpoR.
Figure 8.
No effect of Epo on AKT or STAT5 phosphorylation with NCI-H838 lung carcinoma cell line.
(A) NCI-H838 cells were serum and Epo-starved overnight in medium containing 0.1% serum then treated with the indicated growth factors for the indicated times in the same conditioned minimal medium (37°C) with added growth factors. Blots were probed with specific antibodies to the indicated total or phosphorylated molecules. Data shown using NCI-H838 cells obtained from ATCC. UT-7/Epo and 769-P cells served as positive and negative controls respectively. (B) Cells were starved in 0.0% FBS for 24 Hrs and then rHuEpo, Vehicle, or Growth Factor Cocktail Treatment (HGF, IGF-1 and EGF) was added to quiescent cells. Blots were probed with specific antibodies to the indicated total or phosphorylated molecules. NCI-H838 cells were obtained from T. Lappin University of Dublin, Ireland. UT-7/Epo served as a positive and control.
Figure 9.
No effect of Epo on pAKT, pSTAT3, pS6RP or pSTAT5 with NCI-H838 lung carcinoma cell line by Phosflow.
Cells were grown, treated for 30 minutes with the indicated growth factors and recovered. Fixed and permeabilized cells were washed and stained at room temperature with fluorochrome-conjugated antibodies specific for the phosphorylated (p) forms of STAT5, STAT3 and AKT, and S6 ribosomal (S6RP) proteins. Stained cells were run on a LSRII (FACS) instrument. For both vehicle and Epo treated samples 10,000 events were acquired during the FACS acquisition. Through gating and use of caspase 3 all events were from viable and intact cells. The data in the figure represents a single measurement for each cell line across all measured phospho-proteins. Results are reported as mean fluorescence intensity (fold change) in treated samples compared to vehicle. The dotted line shows the threshold level of staining that can be detected above background. This value represents the minimum fold change relative to vehicle required to accurately define a positive cytokine response signal in the Phosflow assay. The threshold fold change of 1.2 was experimentally validated previously through experiments where stimulated cells were titrated into negative control cells (data not shown). UT-7/Epo cells were the positive control and 769-P and HT-29 cells were negative controls.