Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2008 Apr 4;283(14):9049–9059. doi: 10.1074/jbc.M708022200

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

Copyright © 2008, The American Society for Biochemistry and Molecular Biology, Inc.

PMC Copyright notice

FIGURE 7. — KCa3.1 and KCa2.3 are translocated out of the ER by Derlin-1. A, cells were co-transfected with a short hairpin RNA against Derlin-1 (shDerlin-1) and either wild type (lanes 1 and 2) or E112A (lanes 3 and 4) KCa3.1. Expression of KCa3.1 was evaluated by IB (top panel). Knockdown of Derlin-1, using shDerlin-1, was confirmed by IB (bottom panel). The ratio in the absence and presence of shDerlin-1 is indicated. As is apparent in the top panel, knockdown of Derlin-1 resulted in an increase in both wild type and E112A KCa3.1 expression. 20 μg of total protein was loaded per lane in these experiments. B, co-IP of Derlin-1 with either Myc-tagged wild type KCa3.1 (lanes 1–4) or E112A KCa3.1 (lanes 5–8). Cells were co-transfected with either wild type or E112A Myc-tagged KCa3.1 plus empty vector (lanes 1 and 5), HA-tagged Derlin-1 plus empty vector (lanes 2 and 6), or Myc-tagged KCa3.1 plus HA-tagged Derlin-1 (lanes 3 and 4 and 7 and 8). Lysate was subject to immunoprecipitation (IP) using either an anti-Myc Ab (lanes 1–3 and 5–7) or a control IgG (lanes 4 and 8) and subsequently IB for Derlin-1 using an anti-HA Ab. Derlin-1 was detected by IB in lanes 3 and 7, confirming association between KCa3.1 and Derlin-1. Bottom panel, total lysate (20 μg) was probed for Derlin-1 to confirm equivalent expression. No Derlin-1 was detected in lanes 1 and 5 as these were only transfected with Myc-tagged KCa3.1. C, cells were co-transfected with a short hairpin RNA against Derlin-1 (shDerlin-1) and either wild type (1st and 2nd lanes) or E370A (3rd and 4th lanes) KCa2.3. Expression of KCa2.3 was evaluated by IB (top panel). Knockdown of Derlin-1 using shDerlin-1 was confirmed by IB (bottom panel). The ratio in the absence and presence of shDerlin-1 is indicated. As is apparent in the top panel, knockdown of Derlin-1, using shDerlin-1, resulted in an increase in E370A KCa2.3 expression. 20 μg of total protein was loaded per lane. D, co-IP of Derlin-1 with either wild type KCa2.3 (lanes 1–4) or E370A KCa2.3 (lanes 5–8). Cells were co-transfected with either wild type or E370A KCa2.3 plus empty vector (lanes 1 and 5), HA-tagged Derlin-1 plus empty vector (lanes 2 and 6), or KCa2.3 plus HA-tagged Derlin-1 (lanes 3 and 4 and 7 and 8). Lysate was subject to IP using either an anti-HA Ab (lanes 1–3 and 5–7) or a control IgG (lanes 4 and 8) and subsequently IB for KCa2.3. KCa2.3 was detected by IB in lanes 3 and 7, confirming association between KCa2.3 and Derlin-1. Bottom panel, total lysate (20 μg) was probed for KCa2.3 to confirm equivalent expression. No KCa2.3 was detected in lanes 2 and 6 as these were only transfected with HA-tagged Derlin-1. Note that E370A KCa2.3 is expressed at a significantly lower level than wild type KCa2.3 as expected.