EeLepf

Protocol tips

Upstream tips	Protocol tips	Downstream tips
leptin-HA	Plasmids were introduced into protoplasts by polyethylene glycol-mediated transformation. Protein extracts were prepared at 24 h after transformation or at the indicated time points.	Protein extracts were analyzed by western blotting with anti-HA (Roche Diagnostics, Indianapolis, IN), anti-actin (MP Biomedicals, Solon, OH), anti-GFP (Bio-Application, Pohang, Korea), or anti-BiP antibodies47–49. The protein blots were developed with an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ), and images were obtained using an LAS4000 image analyzer (Fujifilm, Tokyo, Japan). To quantify expression levels, intensity of protein bands in western blot images was measured using Multi-Gauge program equipped to LAS4000 image analyzer (FUJIFILM, Tokyo, Japan). Band intensities were added in cases of glycosylated proteins except the bottom band that was considered as the non-glycosylated form.

Upstream tips
leptin-HA
Protocol tips
Plasmids were introduced into protoplasts by polyethylene glycol-mediated transformation. Protein extracts were prepared at 24 h after transformation or at the indicated time points.
Downstream tips
Protein extracts were analyzed by western blotting with anti-HA (Roche Diagnostics, Indianapolis, IN), anti-actin (MP Biomedicals, Solon, OH), anti-GFP (Bio-Application, Pohang, Korea), or anti-BiP antibodies47–49. The protein blots were developed with an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ), and images were obtained using an LAS4000 image analyzer (Fujifilm, Tokyo, Japan). To quantify expression levels, intensity of protein bands in western blot images was measured using Multi-Gauge program equipped to LAS4000 image analyzer (FUJIFILM, Tokyo, Japan). Band intensities were added in cases of glycosylated proteins except the bottom band that was considered as the non-glycosylated form.

Publication protocol

Plasmids were introduced into protoplasts by polyethylene glycol-mediated transformation47. Protein extracts were prepared at 24 h after transformation or at the indicated time points48. The protoplasts were treated with tunicamycin (10 μg/mL; Sigma-Aldrich, St. Louis, MO) immediately after transformation. Cycloheximide (50 μg/mL; Sigma-Aldrich, St. Louis, MO) treatment was applied at 12 h after transformation. Protein extracts were analyzed by western blotting with anti-HA (Roche Diagnostics, Indianapolis, IN), anti-actin (MP Biomedicals, Solon, OH), anti-GFP (Bio-Application, Pohang, Korea), or anti-BiP antibodies47–49. The protein blots were developed with an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ), and images were obtained using an LAS4000 image analyzer (Fujifilm, Tokyo, Japan). To quantify expression levels, intensity of protein bands in western blot images was measured using Multi-Gauge program equipped to LAS4000 image analyzer (FUJIFILM, Tokyo, Japan). Band intensities were added in cases of glycosylated proteins except the bottom band that was considered as the non-glycosylated form.

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