American Society of Hematology

Figure 5.

$Figure 5. Effect of p19INK4d knockdown on HSP70 expression, localization, and ERK activity. (A) Representative images of western blotting showing HSP70 and HSP27 expression in erythroblasts infected with Lucif shRNA or p19INK4d shRNA (left). Quantitative analysis of protein expression data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. (B) Representative immunofluorescence images showing HSP70 localization in erythroblasts infected with Lucif shRNA or p19INK4d shRNA. DAPI was used to stain the nucleus. (C) Western blotting analysis of nuclear and cytoplasmic fractions of HSP70. RCC1 and α-tubulin were used as nuclear and cytoplasmic markers, respectively. (D) Representative images of western blotting for p-ERK, ERK, p-AKT, and AKT in erythroblasts infected with Lucif shRNA or p19INK4d shRNA (left). Quantitative analysis of western blotting data from 3 independent experiments is shown (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. (E) Representative images of western blotting showing p-ERK, HSP70, GATA1, and KLF1 expression level in erythroblasts treated with 0, 5, or 10 μM FR180204 (left). Quantitative analysis of western blotting data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. (F) Representative immunofluorescence images showing HSP70 localization in erythroblasts treated with 0 or 10 μM FR180204. DAPI was used to stain the nucleus. (G) Western blotting analysis of nuclear and cytoplasmic fractions of HSP70 in erythroblasts from untreated and treated with 10μM FR180204. RCC1 and α-tubulin were used as nuclear and cytoplasmic markers, respectively. Statistical analysis of data from 3 independent experiments and bar plot represents mean ± SD of triplicate samples. *P < .05, **P < .01, ***P < .001.$

Effect of p19^INK4dknockdown on HSP70 expression, localization, and ERK activity. (A) Representative images of western blotting showing HSP70 and HSP27 expression in erythroblasts infected with Lucif shRNA or p19^INK4d shRNA (left). Quantitative analysis of protein expression data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. (B) Representative immunofluorescence images showing HSP70 localization in erythroblasts infected with Lucif shRNA or p19^INK4d shRNA. DAPI was used to stain the nucleus. (C) Western blotting analysis of nuclear and cytoplasmic fractions of HSP70. RCC1 and α-tubulin were used as nuclear and cytoplasmic markers, respectively. (D) Representative images of western blotting for p-ERK, ERK, p-AKT, and AKT in erythroblasts infected with Lucif shRNA or p19^INK4d shRNA (left). Quantitative analysis of western blotting data from 3 independent experiments is shown (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. (E) Representative images of western blotting showing p-ERK, HSP70, GATA1, and KLF1 expression level in erythroblasts treated with 0, 5, or 10 μM FR180204 (left). Quantitative analysis of western blotting data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. (F) Representative immunofluorescence images showing HSP70 localization in erythroblasts treated with 0 or 10 μM FR180204. DAPI was used to stain the nucleus. (G) Western blotting analysis of nuclear and cytoplasmic fractions of HSP70 in erythroblasts from untreated and treated with 10μM FR180204. RCC1 and α-tubulin were used as nuclear and cytoplasmic markers, respectively. Statistical analysis of data from 3 independent experiments and bar plot represents mean ± SD of triplicate samples. *P < .05, **P < .01, ***P < .001.

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