Journal: Journal of Cachexia, Sarcopenia and Muscle
Article Title: Long‐noncoding RNA Atrolnc‐1 promotes muscle wasting in mice with chronic kidney disease) Long‐noncoding RNA Atrolnc‐1 promotes muscle wasting in mice with chronic kidney disease
Figure Lengend Snippet: Atrolnc‐1 regulates MuRF‐1 via interacting with ABIN‐1 in vitro and in vivo . (A) In situ hybridization analysis with a biotin‐labelled Atrolnc‐1 antisense probe in C2C12 cell. Atrolnc‐1 positive signals manifest as brown granules located both in the nuclear (N) and cytoplasm (Cyto). Atrolnc‐1 sense probe was used as negative control (CTL). Scale bar = 20 μm. (B) SDS‐PAGE gels exhibit the results of RNA pull‐down assay. The interaction of biotin‐labelled Atrolnc‐1 probe (marked as 2) with proteins from cytoplasm (left gel) and nucleus (right gel) extracted from C2C12 cells are shown. Atrolnc‐1 antisense probe was used as control (marked as 1). M represented molecular marker. (C) The table lists mass spectrometry result of proteins interacting with Atrolnc‐1. In the gels in (B), ABIN‐1, an inhibitor of NF‐kB signalling, was found to bind with biotin‐labelled Atrolnc‐1 probe in cytoplasmic extracts from myotubes. The iBAQ denotes the sum of all the peptides intensities divided by the number of observable peptides of a protein. (D) Left panel: a representative immunoblotting shows a band at 72 kD using ABIN‐1 antibody in the proteins pulled‐down by biotin‐labelled Atrolnc‐1 probe. Right panel: in cytoplasmic extracts of tibialis anterior (TA) muscle from mice, immunoblotting also detected a band at 72 kD in RNA pull‐down assay by biotin‐labelled Atrolnc‐1 probe. (E) The result of immunoprecipitation (IP) in TA muscle from mice with or without chronic kidney disease (CKD). Ten per cent of the IP product was used to conduct western blot (low panel), showing a band at 72 kD using ABIN‐1 antibody (ABIN‐1 Ab) while a RT‐PCR of Arolnc‐1(upper panel) was performed using the rest of IP product. Notice that the Atrolnc‐1 interacted with ABIN‐1 was increased in CKD mice. Mock, normal mouse IgG. (F) Immunoblot in upper panel shows increased ABIN‐1 expression in C2C12 myotubes after transduced by CRISPR/dCas9 mediated synergistic activation mediator transcription activation system. Bar graph (low panel) illustrates the quantity result (mean ± SEM; n = 5, * P
Article Snippet: To obtain the biotin‐labelled Atrolnc‐1‐RNA probe, linearized pBlue‐Atrolnc‐1 plasmid was incubated with biotin RNA labelling mix (Roche Molecular Systems, Inc. Hague Road, IN) for 20 min at 37°C, in vitro transcription was performed using a MAXIscript® T7/T3 Transcription Kit (Thermo Fisher, Waltham, MA, USA) following manufacturer's instructions.
Techniques: In Vitro, In Vivo, In Situ Hybridization, Negative Control, CTL Assay, SDS Page, Pull Down Assay, Marker, Mass Spectrometry, Mouse Assay, Immunoprecipitation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, CRISPR, Activation Assay