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3 protocols using igg rabbit

1

Antibody Characterization for Cell Biology

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The following are other antibodies used in this study: anti-actin-HRP (horseradish peroxidase) (Santa Cruz, sc-47778), mouse anti-PCNA (Abcam, ab29), rabbit anti-XPO1-C-terminal (Santa Cruz, sc-5595), mouse anti-XPO1-N-terminal (Santa Cruz, sc-136220), mouse anti-Coilin (Sigma C-1862), mouse anti-XPO5 (Abcam ab57491), mouse anti-XPOT (Abcam ab49933), rabbit anti-H3 (Abcam ab1791), mouse anti-GEMIN5 (Santa Cruz, sc-136200), mouse anti-SMN1 (Abcam, ab5831), goat anti-LaminB (Santa Cruz, sc-6216), mouse anti-CSE1L (Santa Cruz sc-135855), rabbit anti-Dyskerin (Santa Cruz sc-48794), rabbit anti-PHAX (Bethyl, A303–916A), goat anti-PHAX (M-19) (Santa Cruz, sc-11704), mouse anti-UBF1 (Santa Cruz sc-13125), mouse anti-nucleolin (Enzo, ADI-KAM-CP100) mouse anti-TRF2 (Imgenex, IG124A), mouse anti-XPO5 (Abcam ab57491), mouse anti-NFKB (Santa Cruz sc-372), mouse anti-RANBP2 (Santa Cruz, sc-74518), mouse anti-Fibrillarin (Abcam, ab4566), mouse anti-FLAG M2 (Sigma), IgG mouse (Santa Cruz sc-2025), mouse anti-Coilin (Sigma, C1862), rabbit anti-TCAB1 (Novus, NB100–68252); IgG rabbit (Abcam ab37415). All Alexa-conjugated secondary antibodies were purchased from Life Technologies.
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2

Protein Expression Analysis of FKRP in Larval Tissues

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Larval heads were removed and used for genotyping, bodies were snap-frozen, homogenised in immunoprecipitation buffer (Pierce). In all, 30 μg of protein was loaded on a 4–12% Bis-Tris Protein Gel MES Buffer (ThermoFisher Scientific). Primary antibodies were prepared in TBS blocking buffer (LI-COR): fkrp: rabbit polyclonal 1:500 fkrp48 (link), 1:500, Beta-Tubulin-Loading Control (Abcam: ab6046), Golgi 50 kda, IgG rabbit (Abcam: ab6046) 1:10,000, incubated in secondary antibody IRDye 800CW goat anti-rabbit (LI-COR) 1:5000. Blots were scanned on  a infrared scanner (Odyssey). Co-immunoprecipitation utilised 10ug of antibody, fibronectin or FKRP, and a protein-G dynabead kit (Thermofisher).
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3

Collagen 1a Protein Quantification

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Larvae were homogenized in immunoprecipitation buffer (Pierce, Rockford, Illinois), and 30 μg of protein with was loaded on a 3-8% Tris-Acetate Protein Gel (ThermoFisher Scientific, Waltham, Massachusetts). Primary antibodies were made up in same Licor TBS blocking buffer: Collagen 1a, IgG rabbit (AbCam, United Kingdom: ab2370) 1/500, Beta-Tubulin Loading Control 50 kDa, IgG rabbit (AbCam, United Kingdom: ab6046) 1/10 000 and incubated in secondary antibody IRDye 800CW goat anti rabbit (LI-COR) 1/5000. Blots were scanned on the Odyssey scanner.
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