Anti myc tag antibody

Cells harboring the promoters were induced as described above and harvested. Cells were washed once in PBS containing 2% FBS and stained using anti Myc tag antibody (Millipore) and incubated on ice for 1 h. Cells were washed once and stained using a secondary anti mouse APC (Jackson) and incubated on ice for 30 min. Cells were washed twice and suspended with DAPI 1 µg/mL and FACS were measured using a Beckman Coulter^® Cytoflex™ flow cytometer.

HeLa cells were seeded on 12 mm circular glass coverslips and transiently co-transfected with Asc1/CD98hc as previously described. 24 h after transfection the slides were washed with PBS and then incubated with 2.5 μg ml⁻¹ of Texas Red-X-labeled wheat germ agglutinin (Thermo Fisher Scientific) at room temperature for 10 min. The cells were subsequently washed with PBS and fixed with 4% paraformaldehyde in PBS at room temperature for 20 min. The fixed cells were then permeabilized with a solution of 0.1% Triton X-100, 1% BSA in PBS at room temperature for 30 min. Afterwards the coverslips were incubated with a 1:500 dilution of anti-Myc-tag antibody (05–724, Millipore) in 1% BSA in PBS at room temperature for 1 h, followed by washes with PBS and a second incubation with a secondary Alexa Flour 488-labeled anti-mouse IgG antibody (A11029, Thermo Fisher Scientific) in 1% BSA in PBS at room temperature for 1 h. Nuclear staining was performed with a 1:20,000 dilution of Hoechst 33342 (Thermo Fisher Scientific) in PBS at room temperature for 20 min. Coverslips were then mounted using Fluoromount^TM aqueous mounting medium (Thermo Fisher Scientific) and imaged using a Leica TCS SP5 Spectral Confocal Multiphoton system.

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.