Figure 3
Figure 3. PDGF-BB mediates downstream target AKT/ERK pathway activation via PI3K and SFK pathways in leukemic LGLs. (A) Western blot assay was carried out for SFK protein expression in PBMCs from 6 patients with T-LGL leukemia (LGL) and 6 normal controls (NL). The gels in the bottom panel indicate SFK expression of 60 KDa molecular weight. Kinase assay (top panel) was performed in IP samples to determine SFK activities (enolase). (B-C) Phospho-AKT and phospho-ERK (p44/42 MAPK) expression was determined in PBMC lysates from a representative patient with T-LGL leukemia (B) and a patient with NK-LGL leukemia(C) using Western blot assay. Western blot assay for GAPDH expression was performed to confirm equal loading of total protein in each lane. (D-G) Densitometry analysis was performed on Western blot results from 3 patients with T-LGL leukemia (D,F) and 3 patients with NK-LGL leukemia (E,G) to determine the average level of phospho-AKT expression (D-E) or phospho-ERK expression (F-G). Data expressed as relative expression units of p-AKT/GAPDH or p-ERK/GAPDH ratios. (H) Western blot assay was performed for phospho-AKT protein expression in lysates of CD8+ cells from a representative patient with T-LGL leukemia. Cells received different treatments as indicated. (Lane 1) Medium only. (Lane 2) Ten percent pooled sera from 3 patients with T-LGL leukemia. (Lanes 3,6) Ten percent pooled sera from either T-LGL leukemia patients or normal controls that received 2-hour preincubation with anti–PDGF-BB neutralizing antibody. (Lane 4) Ten percent pooled sera from patients with T-LGL leukemia that received 2-hour preincubation with IgG isotype control antibody. (Lane 5) Ten percent pooled sera from 3 normal controls. Western blot analysis for GAPDH was performed to confirm equal loading of total protein in each lane. (I) Densitometry was performed for Western blot results from 3 different experiments to determine the average level of phospho-AKT protein expression. *P < .03, no treatment control (NTC) versus 10% T-LGL patient sera treatment; **P < .05, treatment with 10% patient sera versus treatment with 10% patient sera preincubated with anti–PDGF-BB neutralizing antibody.

PDGF-BB mediates downstream target AKT/ERK pathway activation via PI3K and SFK pathways in leukemic LGLs. (A) Western blot assay was carried out for SFK protein expression in PBMCs from 6 patients with T-LGL leukemia (LGL) and 6 normal controls (NL). The gels in the bottom panel indicate SFK expression of 60 KDa molecular weight. Kinase assay (top panel) was performed in IP samples to determine SFK activities (enolase). (B-C) Phospho-AKT and phospho-ERK (p44/42 MAPK) expression was determined in PBMC lysates from a representative patient with T-LGL leukemia (B) and a patient with NK-LGL leukemia(C) using Western blot assay. Western blot assay for GAPDH expression was performed to confirm equal loading of total protein in each lane. (D-G) Densitometry analysis was performed on Western blot results from 3 patients with T-LGL leukemia (D,F) and 3 patients with NK-LGL leukemia (E,G) to determine the average level of phospho-AKT expression (D-E) or phospho-ERK expression (F-G). Data expressed as relative expression units of p-AKT/GAPDH or p-ERK/GAPDH ratios. (H) Western blot assay was performed for phospho-AKT protein expression in lysates of CD8+ cells from a representative patient with T-LGL leukemia. Cells received different treatments as indicated. (Lane 1) Medium only. (Lane 2) Ten percent pooled sera from 3 patients with T-LGL leukemia. (Lanes 3,6) Ten percent pooled sera from either T-LGL leukemia patients or normal controls that received 2-hour preincubation with anti–PDGF-BB neutralizing antibody. (Lane 4) Ten percent pooled sera from patients with T-LGL leukemia that received 2-hour preincubation with IgG isotype control antibody. (Lane 5) Ten percent pooled sera from 3 normal controls. Western blot analysis for GAPDH was performed to confirm equal loading of total protein in each lane. (I) Densitometry was performed for Western blot results from 3 different experiments to determine the average level of phospho-AKT protein expression. *P < .03, no treatment control (NTC) versus 10% T-LGL patient sera treatment; **P < .05, treatment with 10% patient sera versus treatment with 10% patient sera preincubated with anti–PDGF-BB neutralizing antibody.

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