Sterile slides were inserted into 24-well plates; pretransfected HeLa cells were plated into each well at a concentration of 5 × 104/ml and incubated at 37°C with 5% CO2 for 24 h. Next, 4% paraformaldehyde was added for 10 min at room temperature for fixation, permeabilized with 0.25% Triton X-100. Then, cells were washed with PBS, three times for 1 min each time, and blocked with 1% BSA for 1 h at room temperature. Subsequently, anti-REGγ and anti-HBD antibodies were incubated at 4°C overnight. Cells were incubated with secondary antibodies Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG (Abmart) for 1 h, and nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Images of the cells were captured under fluorescence microscopes (LI-COR Bioscience Co.) and analyzed by Image-Pro Plus 6.0 software.
Image pro plus 6
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2 protocols using image pro plus 6
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Immunofluorescence Staining of REGγ and HBD in HeLa Cells
2
Quantitative Protein Analysis by Western Blot
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Western blotting was performed according to our previous study [28] (link). 25μg protein was separated on 12.5% SDS-polyacrylamide gels. After SDS-PAGE, the proteins were transferred to a PVDF membrane, and then blocked with 8% (w/v) milk for 1 h. The membrane was incubated with antibodies against Adrp (15294-1-AP; ProteinTech, Chicago, Illinois, USA), Gm130 (ab30637; Abcam, Cambridge, MA, USA), Calnexin (ab10286), Cox IV (ab33985), Gapdh (ab9485), Atgl (ab99532), Plin5 (ab222811), Creb (ab31387), Pgc1α (ab84139), Rab32 (ab229604), Cd36 (ab64014), Dgat1 (ab54037), Cox-1 (ab109025) and Mfn-1 (ab221661) overnight at 4 °C, and then processed with goat anti-rabbit IRDye 800CW secondary antibody, (926-32211; Li-Cor Biosciences, Lincoln, NE, USA). The protein bands were visualized with an Odyssey Infrared Fluorescent Western Blots Imaging System (Li-Cor Bioscience) and quantified by Image-Pro Plus 6.0.
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