Argonaute CLIP defines a deregulated miR-122 bound transcriptome that correlates with patient survival in human liver cancer - Raw autoradiograms and Western Blots

2019-07-19T08:06:17Z (GMT) by Joseph Luna
JL0109 - Autoradiogram of 32P-labelled RNA crosslinked to IP purified AGO from five normal adjacent (left) or matched HCC tumor from from livers of human HCC patients (right) using the 2A8 anti-Ago antibody, exposed overnight after running on an 8% Novex NuPage gel. Irrelevant antibody control “IgG”, non-crosslinked control "-XL", and high “Hi” RNAse A control are shown. RNA-protein complexes from 110-150kDa were used for subsequent RNA sequencing library preparation. A weak band of ~110kDa is typically observed under non-crosslinked conditions. Protein size markers labeled with boxes at indicated molecular weights. Mass of sample (in milligrams) are indicated beneath each lane. JL0173 - Autoradiogram of 32P-labelled RNA crosslinked to IP purified AGO from four 122KO (top) or five WT floxed control livers (bottom) using the 2A8 anti-Ago antibody, exposed overnight after running on an 8% Novex NuPage gel. Irrelevant antibody control “IgG”, non-crosslinked control "-XL", and high “Hi” RNAse A control are shown. RNA-protein complexes from 110-150kDa were used for subsequent RNA sequencing library preparation. A weak band of ~110kDa was typically observed under non-crosslinked conditions. Protein size markers labeled with boxes at indicated molecular weights. Mass of sample (in milligrams) and sex of animal are indicated beneath each lane. Fig 7A - FLAG-tagged human BCL9 expression levels in 293T cells after transfection with 50nM control or miR-122 mimic. WT BCL9 is compared to a mutant where all 6 exonic miR-122 sites were mutated while preserving amino acid sequence. Blots were imaged on a LiCor with ImageStudio Software V5.2. Short exposure displays Flag BCL9 channel (800nm) and GAPDH channel (680nm). Long exposure shows only the flag-BCL9 channel signal (800nm). Fig 7B - FLAG-tagged human BCL9 expression levels in MHCC-LM3 cells after transfection with 50nM control or miR-122 mimic. WT BCL9 is compared to a mutant where all 6 exonic miR-122 sites were mutated while preserving amino acid sequence. Blots were imaged on a LiCor with ImageStudio Software V5.2. Short exposure used for GAPDH normalization (lower set of bands). Long exposure shows only the flag-BCL9 channel signal in the upper set of bands.