Autophagy Forum

Company: Abgent

Catalog #: AP3301a

Comments:

The antibody to LC3 phosphorylated at S12 recognizes rat LC3 as well as LC3 in human cell lines and mouse primary neurons, when used at 1/500 for western blot. Phosphorylation is decreased upon autophagy induction.

Reference: http://jcb.rupress.org/content/early/2010/08/11/jcb.201002108.full

Cherra SJ, 3rd, Kulich SM, Uechi G, Balasubramani M, Mountzouris J, Day BW, Chu CT: Regulation of the autophagy protein LC3 by phosphorylation, J Cell Biol 190:533-539

In primary neurons, the band co-migrates with LC3-I. In tumor cell lines, there were additional slightly higher molecular weight bands, and in some tumor cells the ~20 kDa band predominates. An antibody to a C-terminal region of LC3 (AP1802b, 1/500) recognizes the band(s) recognized by phospho-LC3. It is unclear whether the phosphorylation affects affinity for N-terminal antibodies for LC3.

As the Journal of Cell Biology editorial policy is to remove catalog numbers from Methods, I have appended a table of antibodies used.

Antibody Cat # Company Dilution Assay
Cleaved-LC3 AP1806a Abgent 1:250 IF
LC3 Nterm (2G6) 0260-100Nanotools …1:500 WB, cell lines
…1:1000 or more WB, 1° neurons
LC3 Cterm AP1802b Abgent 1:500 WB
pLC3 S12 AP3301a Abgent 1:500 WB
pLC3 T12 AP3530a Abgent 1:500 WB
GFP A6455 Invitrogen 1:5000 IF & WB
HA (16B12) MMS-101R Covance 1:1000 IF
GAPDH Ab37168 Abcam 1:10,000 WB
Lamin A/C 2032 Cell signalling 1:1000 WB
β-Actin A5316 Sigma 1:5000 WB
Atg7600-401-487 Rockland 1:1000 WB

Posted by: Charleen T. Chu

Institution: University of Pittsburgh School of Medicine

This entry was posted on Wednesday, September 29th, 2010 at 4:06 pm and is filed under Reagents. You can follow any responses to this entry through the RSS 2.0 feed. You can leave a response, or trackback from your own site.