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. 2018 Jun 19;20(5):511–517. doi: 10.4103/aja.aja_36_18

Figure 1.

Figure 1

LOC283070 directly interacts with PHB2. (a) RNA pull-down experiment in LNCaP-AI cells. Proteins from LNCaP-AI extracts were pulled down with the biotin-RNAs, subjected to SDS-PAGE, and visualized by silver staining. The band, which was specific to LOC283070 in LNCaP-AI but not in two controls, was pointed out by the arrow and subjected to mass spectrometry. (b) Mass spectrometry identification of target band of PHB2. (c) Proteins pulled down by the biotin-RNAs as in (a) were analyzed by western blotting with PHB2 antibody. (d) RIP assay was performed using PHB2 antibody and was validated by agarose electrophoresis using LOC283070 primers. Lane mark: DNA molecular marker, lane: 1, 3, 5, 7 from LNCaP cells and 2, 4, 6, 8 from LNCaP-AI cells. RNA extracted with SNRP70 antibody and IgG were amplified by PCR experiments using U1 primers (as positive and negative controls, respectively). (e) Fold change enrichment of LOC283070 in LNCaP and LNCaP-AI was calculated comparing with the input in panel in qRT-PCR assay (n = 3, **P < 0.01). (f) PHB2 protein expression measured by western blotting. PHB2: prohibitin 2; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; LNCaP-AI: androgen-independent LNCaP; RIP: RNA-binding protein immunoprecipitation.