LC3B antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For Immunoblot dilute Ab at overnight at 4°C in 1:500 dilution. - For IF dilute Primary Ab dilution-1:200 and incubate for 1h

Protocol tips
- For Immunoblot dilute Ab at overnight at 4°C in 1:500 dilution. - For IF dilute Primary Ab dilution-1:200 and incubate for 1h

Publication protocol

Immunoblot
Protein lysates (40 μg) were separated by SDS-PAGE, transferred to nitrocellulose membrane, blocked for 1 hour in Tris-buffered saline/0.1% Tween-20/5% nonfat milk, then incubated overnight at 4°C in 1:500 rabbit anti-Atg12 or rabbit anti-LC3B (both from Cell Signaling Technology) in Tris-buffered saline/0.1% Tween-20/5% BSA. The membrane was washed, probed with polyclonal sheep anti–rabbit conjugated to horseradish peroxidase, and protein bands were detected using Enhanced Chemiluminescence Substrate (Western Lighting Plus ECL; PerkinElmer Life and Analytical Sciences). The membrane was stripped and reprobed with monoclonal mouse anti-GAPDH (clone 6C5; Abcam).

Immunofluorescence microscopy
Cytospins of T2.B35 pUL138-eGFP stable transfectants were air dried, fixed with 4% paraformaldehyde, pH 7.4, for 20 minutes at room temperature, washed twice with PBS, and extracted with 0.5% Triton X-100 for 15 minutes. Slides were blocked for 30 minutes with PBS/5% normal goat serum/5% FCS/3% BSA, then incubated for 1 hour in 1:200 polyclonal rabbit anti–human LC3 (Cell Signaling Technology) and 1:200 monoclonal mouse anti-GFP (ab1218; Abcam), or 1:100 monoclonal mouse anti–human LAMP2 (clone H4B4; Abcam) and 1:300 polyclonal rabbit anti-GFP (ab6556, Abcam) in blocking buffer. Slides were washed 3 times in blocking buffer, incubated with 1:200 goat anti–rabbit or anti–mouse conjugated to AlexaFluor-546 or AlexaFluor-488 (Invitrogen) for 40 minutes, washed 3 times, and mounted with ProLong Gold reagent with 4,6-diamidino-2-phenylindole (Invitrogen). For studies on surface MHC class I internalization, T2.B35 pUL138-eGFP stable transfectants were incubated in 1 μg/mL mouse anti–human MHC class I (clone W6/32) or mouse IgG2aK isotype control (clone eBM2a, eBioscience) in complete medium at 37°C 6% CO2 for 4 hours, washed, then air dried, fixed, extracted, blocked, and stained with goat anti–mouse conjugated to AlexaFluor-546. For colocalization of internalized MHC class I with LC3, nontransfected T2.B35 cells were incubated in the above antibodies with or without 80μM chloroquine for 60 hours. Cells were imaged on an Olympus IX70 microscope using DeltaVision microscopy system, and image deconvolution and colocalization analyses were performed using softWoRx (both from Applied Precision). A minimum of 50 cells were analyzed.

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