Rabbit anti flag m2 antibody

To quantify cell surface receptor expression, HEK293 (ThermoFisher Scientific, Nr. R70507) cells transfected with G-CASE and pcDNA or N-terminally FLAG-tagged H_1/2R constructs were grown for 48 h in transparent 96-well plates (Brand, Wertheim, Germany) and washed once with 0.5% BSA (Merck KGaA, Darmstadt, Germany) in PBS. Next, cells were incubated with a rabbit anti-FLAG M2 antibody (142 ng/mL) (Cell Signaling Technology, Danvers, MA, USA) in 1% BSA–PBS for 1 h at 4 °C. Following incubation, the cells were washed three times with 0.5% BSA–PBS and incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody (30 ng/ml) (Cell Signaling Technology, Danvers, MA, USA) in 1% BSA–PBS for 1 h at 4 °C. The cells were washed three times with 0.5% BSA/PBS, and 50 µl of the 3, 3′, 5, 5′ tetramethyl benzidine (TMB) substrate (BioLegend, San Diego, CA, USA) was added. Subsequently, the cells were incubated for 30 min and 50 µl of 2 M HCl was added. The absorbance was read at 450 nm using a BMG ClarioStar Plus plate reader.

Polyclonal galectin-12 antibodies were generated in galectin-12 knockout mice [17 (link)]. Mouse anti-FLAG M2 antibody and Anti-FLAG M2-agarose were purchased from Sigma. They were used for Western blotting and immunoprecipitation of Flag-tagged proteins. For immunofluorescence, rabbit anti-Flag M2 antibody (Cell Signaling Technology) was used. Mouse anti-LAMP1 and anti-Myc tag antibodies were from Millipore and Syd Labs, respectively. MitoTracker Deep Red, rabbit anti-LAMP1 and anti-Myc tag antibodies were purchased from Cell Signaling Technology. Rabbit anti-perilipin-1 was from Affinity Bioreagents. Rabbit anti-calnexin was purchased from Stressgen. The following cell lines were purchased from ATCC: mouse fibroblast cell line 3T3-L1 (ATCC CL-173), human embryonic kidney cell line 293T (ATCC CRL-11268), and the human cervical carcinoma cell line HeLa (ATCC CCL-2). They were cultured following standard conditions in DMEM/10% FBS at 5% CO₂, 37°C.

Immunofluorescent staining was performed for both 12 hpf and 24 hpf embryos after the TUNEL assay. Embryos were washed using PBS Tween 20 (1% PBT) three times for 5 min. Embryos were pre-incubated in 0.75 ml blocking buffer (1% DMSO and 5% sheep serum in PBT) for at least 2 h at room temperature. The pre-blocking solution was removed and mouse anti-8-oxoG (Sigma-Aldrich, Ref. No.: MAB3560, dilution 1:200), rabbit anti-BG4 (absolute antibody, Ref. No.: Ab00174-30.126, dilution 1:200) and rabbit anti- FLAG M2 Antibody (Cell Signaling, Ref. No.: 14793, dilution 1:200), primary antibodies were added in 0.75 ml blocking buffer and incubated at 4°C overnight on a shaker incubator. Unbound primary antibody was washed using PBS Tween 20 (1% PBT) three times for 30 min and secondary antibodies, goat anti-rabbit Alexa Fluor 488 (1:500, Invitrogen Molecular probes), goat anti-mouse Alexa Fluor 488 (1:500, Invitrogen Molecular probes) and goat anti-mouse Alexa Fluor 633 (1:500, Invitrogen Molecular probes) were added in 0.75 ml blocking buffer and incubated at 4°C overnight on a shaker incubator. Unbound secondary antibody was washed using PBS Tween 20 (1% PBT) three times for 30 min and 4′,6-diamidino-2-phenylindole (DAPI) was used to visualize the nuclei. Stained embryos were kept in 70% glycerol. Images were taken using a Nikon confocal microscope.