rabbit anti-grap antibody  (Thermo Fisher)

Journal: The Journal of Neuroscience

Article Title: CACHD1 is an α2δ-Like Protein That Modulates Ca V 3 Voltage-Gated Calcium Channel Activity

doi: 10.1523/JNEUROSCI.3572-15.2018

Figure Lengend Snippet: Characterization of CACHD1 and its effects on CaV3.1 expression. HEK cells were transfected with empty vector (VC), CACHD1, Myc-CACHD1, and GFP-CaV3.1-HA alone or in combination, as shown in each panel. A, HEK cell lysates were analyzed by Western blotting (WB). An antibody to CACHD1 recognized a single protein similar to the predicted size for CACHD1, but also recognized a nonspecific protein in all lysates. B, Cell-surface proteins were biotinylated, and pull-downs were analyzed for CACHD1 and Na+/K+-ATPase (loading control). In control cells, no immunoreactive CACHD1 was detected, confirming antibody specificity. In CACHD1-expressing cells, immunoreactive CACHD1 was detected. In both cell types, immunoreactive Na+/K+-ATPase was detected. C, Cell-surface proteins were biotinylated, and pull-downs were analyzed for GFP-CaV3.1-HA (HA) and Na+/K+-ATPase (loading control). In control cells and cells only expressing CACHD1, no HA signals were detected, confirming antibody specificity. In cells expressing GFP-CaV3.1-HA, HA signals were readily detected. Quantification of the HA signals (normalized to Na+/K+-ATPase) revealed the expression of CACHD1-increased signals for GFP-CaV3.1-HA at the cell surface, *p < 0.05. Na+/K+-ATPase signals were detected in all cell types. D, Inputs of the biotin pull-down assays were analyzed by WB. Signals for HA were detected only in cells expressing GFP-CaV3.1-HA, signals for CACHD1 were detected only in cells expressing Myc-CACHD1, and signals for β-actin were detected in all cell types. All blots are representative of n ≥ 3 experiments.

Article Snippet: The following antibodies were used: mouse anti-HA.11 (clone 16B12, Lot No. B220767, Cambridge Bioscience; RRID: AB_10063630 ); rabbit anti-Na + /K + -ATPase (NB100-80005, Lot No. YH02206, Novus Biologicals; RRID: AB_2063297 ); mouse anti-c-Myc (clone 9E10, Lot No. 087M4765V, catalog #M4439, Sigma-Aldrich; RRID: AB_439694 ); rabbit anti-c-Myc (Lot No. 016M4762V, catalog #C3956, Sigma-Aldrich; RRID: AB_439680 ); mouse anti-β-actin (Lot No. 028K4826, catalog #A5441, Sigma-Aldrich; RRID: AB_476744 ); rabbit anti-CACHD1 (Lot No. QC22258, catalog #AV49592, Sigma-Aldrich; RRID: AB_1852421 ); goat anti-mouse or rabbit IgG coupled to horseradish peroxidase (Stratech Scientific); donkey anti-mouse or rabbit coupled to Alexa Fluor 488, 555, or 647 (Invitrogen). (Note: we experienced vial-to-vial variation with the rabbit anti-CACHD1 antibody for Western blotting during this study.)

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Control

Journal:

Article Title: Intracellular Lipid Droplets Contain Dynamic Pools of Sphingomyelin: ADRP Binds Phospholipids with High Affinity

doi: 10.1007/s11745-010-3424-1

Figure Lengend Snippet: Western blot analysis of lipid droplet proteins isolated from L-cell fibroblasts. Western blots on cell homogenate (lane 1) and lipid droplets (lane 2) were probed with the following affinity-purified antibodies as described in the “Materials and Methods” section: a anti-ADRP, b anti-caveolin-1, c anti-caveolin-2, d anti-Na+, K+-ATPase, e transferrin, and f anti-cytochrome c oxidase

Article Snippet: Rabbit anti-human caveolin-2 (cat. no. NB100-79911) and rabbit anti-human Na + K + -ATPase (cat. no. NB100-80005) were purchased from Novus Biologicals (Littleton, CO).

Techniques: Western Blot, Isolation, Affinity Purification