Ab183094

Muscle biopsy was performed in all patients and specimens were well preserved at −80°C until use. Frozen tissues were sliced into 7 um sections for histological staining, immunohistochemistry (IHC) and immunofluorescence (IF) staining according to standard procedures as reported previously (13 (link)). The following primary antibodies were used to recognize: MuRF-1 (1:50, ab183094, Abcam), complement C5b-9/MAC (1:50, M0777, DAKO) and neural cell adhesion molecule (NCAM)/CD56 (1:50, ab6123, Abcam).

The samples for western blot were prepared by homogenizing the soleus and gastrocnemius muscles in radioimmunoprecipitation assay buffer supplemented with protease inhibitors, followed by sonication and centrifuged at 15,000 rpm for 20 min at 4°C to obtain the supernatant. The protein concentration in the supernatant was measured by a bicinchoninic acid assay kit (Pierce, Rockford, IL, United States). Equal amounts of protein (30 μg) from each sample were electrophoresed on 8%–12% sodium dodecyl sulfate polyacrylamide gel and transferred onto polyvinylidene difluoride membrane. The membranes were first blocked with non-fat milk (5%) for 1 h at RT before incubating with primary antibodies against GAPDH (1:5000, 60004-1-Ig, Protein tech), Atrogin-1 (1:1000, ab168375, Abcam), MuRF1 (1:1000, ab183094, Abcam), P-PI3K (1:1000, ab182651, Abcam), P-AKT (1:1000, Cell signaling Technology), and PI3K (1:1000, ab191606, Abcam). Membranes were washed with Tris-buffered saline containing 0.1% Tween 20 Detergent, followed by a reaction with respective secondary antibodies for 45–60 min. The immunoblots were developed with enhanced chemiluminescence plus reagent, and the results were quantified with laboratory image version 2.7.1.

Tissues or cells were lysed with RIPA buffer. Protein content in lysates was measured using bicinchoninic acid and equal amounts of proteins (20 μg) were separated using SDS-PAGE with β-Actin as a loading control. Separated proteins were transferred to PVDF membranes (Millipore, USA) and subsequently blocked with 5% nonfat dry milk. The membranes were incubated overnight at 4 °C with primary antibodies. The following primary antibodies were used: CPNE1 (1:1000, abcam, ab155675), ATROGIN1 (1:1000, Abcam, ab168372), MuRF1 (1:1000, Abcam, ab183094), MyoG (1:1000, Abcam, ab124800), MyoD (1:1000, Abcam, ab133627), GLB1 (1:1000, Abcam, ab203749), p-eIF2α (1:1000, Cell Signaling Technologies, #9721), eIF2α (1:1000, Abcam, ab169528), p-PERK (1:1000, Cell Signaling Technologies, #3179), PERK (1:1000, Abcam, ab229912), ATF4 (1:1000, Abcam, ab31390) and β-Actin (1:1000, Abcam, ab8226). Protein gray values were measured using Image J software.