Mouse monoclonal anti glyceraldehyde 3 phosphate dehydrogenase gapdh

For Western blot analyses, retina protein samples (25 μg each) in lysis buffer (50 mM Tris-formic acid, 150 mM NaCl, 0.5% sodium deoxycholate, 2% SDS, 2% NP-40, pH8.0) were first separated by SDS-PAGE using standard methodology with 10% polyacrylamide gels (Invitrogen). Proteins then were electrophoretically transferred to polyvinylidene difluoride membranes (Invitrogen). The membrane was blocked for 1 h with Western blocking solution (Invitrogen) and sequentially incubated with a primary antibody overnight at 4 °C followed by an appropriate secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Inc., Dallas, TX) for 1 h. The positive immunoreactions were detected with X-ray film by chemiluminescence using an ECL Western blotting kit (Pierce, Rockford, IL) and developed with a Kodak X-OMAT 2000A Processor. The primary antibodies used in this study were as follows: mouse monoclonal anti-GFAP (1:400; SigmaAldrich); rabbit polyclonal anti-signal transducer and activator of transcription 1 (Stat1 p84/p91) (1:500; Santa Cruz Biotechnology); and mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000; Santa Cruz Biotechnology).

mouse monoclonal anti-GFP at ½000 (cat. no sc-9996, Santa Cruz Biotechnology), mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (cat. no G8795, Gillingham), and mouse monoclonal histone H3 (cat. no 96C10, New England Biolabs) were used for detecting lysate, soluble, and insoluble fractions on Western blot from NP-40 insolubility assays. In the annexin A11/EF-hand protein (calcyclin, ALG-2, and sorcin) binding assays, mouse monoclonal anti-FLAG (cat. no. F3165, Sigma-Aldrich) was used to immunoprecipitate FLAG-tagged EF-hand proteins, and mouse monoclonal anti-GFP (cat. no. sc-9996, Santa Cruz Biotechnology) and mouse monoclonal anti-FLAG or rabbit polyclonal anti-FLAG (cat. no. F7425, Sigma-Aldrich) were used to detect GFP-fused proteins and FLAG-tagged proteins, respectively. Polyclonal rabbit anti-ANXA11 (cat. no. 10479–2-AP, Proteintech) was used for ANXA11^R235Q staining of HEK cells (fig. S4) and spinal cord of the p.D40G SALS patient, SALS patient devoid of known ALS causing mutation, other neurodegenerative disorders, and controls. Polyclonal rabbit anticalcyclin (cat. no. 10245–1-AP, Proteintech) was used for immunohistochemistry (IHC) of patient and control postmortem tissue and detection of FLAG-tagged calcyclin by Western blot in HEK cells.