![Figure 4. Downregulation of nucleolin removes surface nucleolin and eradicates the nucleolin-Fas complex. A nucleolin-specific (906), nontargeting (nonsilencing [NS]) control, and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) targeting short hairpin RNA miR30 constructs were used for transfection of BJAB cells to create a pooled NS control, GAPDH control, and nucleolin pKO (906P1) cell line. Four single-cell clones (906S1, 906S2, 906S4, 906S5) were derived from the original pooled cell line 906P1. (A) Whole-cell lysates were analyzed by IB for nucleolin and Fas protein expression. β-actin was used as a loading control. (B) Densitometry analysis revealed a minimum of 50% knockdown of nucleolin in the pKO cells compared with parental BJAB cells, nonsilencing controls, and GAPDH controls (906P1: P < .026; 906S1: P < .035; 906S2: P < .027; 906S4: P < .013; 906S5: P < .0114). Mean value and standard error of the mean of 3 independent experiments are shown. (C) Nucleolin pKO cells, parental BJABs, and nonsilencing controls were analyzed for nucleolin mRNA levels and normalized to GAPDH (906P1: P < .031, 906S1: P < .095, 906S2: P < .038, 906S4: P < .026, 906S5: P < .038). (D) Surface levels of nucleolin and Fas were analyzed in the parental BJAB, NS control, and nucleolin pKOs by biotinylation followed by strepavidin agarose IP. Histone 3 IB was used as a control for purity of the surface fraction. BJAB whole-cell extracts were used as a positive control for antibody specificity. Input levels of nucleolin and β-actin were used as a loading control. Representative data from 3 different experiments are shown. (E) Association of nucleolin and Fas was analyzed in parental BJAB cells, a NS control, a GAPDH control, and pKOs of nucleolin 906P1, 906S1, 906S2, 906S4, and 906S5 by IP with Fas (B-10) agarose beads. Nucleolin was detected by IB. Mouse isotype-matched IgG and protein G agarose were used as a negative control for nonspecific binding. BJAB whole-cell extracts were used as a positive protein control. Representative data from 3 different experiments are shown.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/121/23/10.1182_blood-2012-12-471094/4/4729f4.jpeg?Expires=1724823324&Signature=2r5bYx90GKGEgGDdU3eh3~~Dxst~9JBN9yi5OnF5Cl3Nq-tZAgXQIQN8-todhxbIk3o6MRiPK~G3SH~plDvBtgjvgGNA5fNQQhGzkk9oHibuGvJY2Jq78QCEnh-DBi1FluJA1lD6GceuhI6WEToIuB-420BQWAd3pJRBgB0rMzCNS3BVczJzFEwhonDlBrF5NpBGxGasyVP9Cemr1I9tXqOXyUd8rKL0oor6Ai3duCy~D3wZWGCkT-8QjQPsDIzDPDLTLGZg~xAE4T~SlicDFJ-HsyQqR6h~s0IKkg1WpjTbEL~U5E-tACnKA~9NjjBgT-JfTwStTOe1EJ07NxkdGQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Downregulation of nucleolin removes surface nucleolin and eradicates the nucleolin-Fas complex. A nucleolin-specific (906), nontargeting (nonsilencing [NS]) control, and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) targeting short hairpin RNA miR30 constructs were used for transfection of BJAB cells to create a pooled NS control, GAPDH control, and nucleolin pKO (906P1) cell line. Four single-cell clones (906S1, 906S2, 906S4, 906S5) were derived from the original pooled cell line 906P1. (A) Whole-cell lysates were analyzed by IB for nucleolin and Fas protein expression. β-actin was used as a loading control. (B) Densitometry analysis revealed a minimum of 50% knockdown of nucleolin in the pKO cells compared with parental BJAB cells, nonsilencing controls, and GAPDH controls (906P1: P < .026; 906S1: P < .035; 906S2: P < .027; 906S4: P < .013; 906S5: P < .0114). Mean value and standard error of the mean of 3 independent experiments are shown. (C) Nucleolin pKO cells, parental BJABs, and nonsilencing controls were analyzed for nucleolin mRNA levels and normalized to GAPDH (906P1: P < .031, 906S1: P < .095, 906S2: P < .038, 906S4: P < .026, 906S5: P < .038). (D) Surface levels of nucleolin and Fas were analyzed in the parental BJAB, NS control, and nucleolin pKOs by biotinylation followed by strepavidin agarose IP. Histone 3 IB was used as a control for purity of the surface fraction. BJAB whole-cell extracts were used as a positive control for antibody specificity. Input levels of nucleolin and β-actin were used as a loading control. Representative data from 3 different experiments are shown. (E) Association of nucleolin and Fas was analyzed in parental BJAB cells, a NS control, a GAPDH control, and pKOs of nucleolin 906P1, 906S1, 906S2, 906S4, and 906S5 by IP with Fas (B-10) agarose beads. Nucleolin was detected by IB. Mouse isotype-matched IgG and protein G agarose were used as a negative control for nonspecific binding. BJAB whole-cell extracts were used as a positive protein control. Representative data from 3 different experiments are shown.
