LC3A/B (D3U4C) XP® Rabbit mAb #12741

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	1:1000 dilution for primary antibody

Protocol tips
1:1000 dilution for primary antibody

Publication protocol

Primary macrophages were harvested and sonicated at 4 °C in RIPA Lysate buffer (50 mm Tris‐HCl (pH: 7.4), 150 mm NaCl, 1% NP‐40, 0.5% Na‐deoxycholate, 0.1% SDS, and protease inhibitor). After quantification with BCA (Thermo scientific, Rockford, IL, USA), 10 μg of the lysate was separated by Novex® NuPAGE® SDS‐PAGE Gel System (Life technologies) and transferred to iBlot PVDF membranes (Life technologies). Blots were blocked in 1:1 PBS, Odyssey Blocking Buffer (LI‐COR bioscience, Lincoln, NE) for 1 h at RT. The blocked membrane was incubated in blocking buffer containing 0.1% Tween‐20 and the respective primary antibodies LC3A/B (Cell signaling, Danvers, MA, USA, 1/1000), Atg7 (Cell signaling, 1:1000), histone H3 (Cell signaling, 4499, 1:2000), VPS34 (Sigma, V9764, 1:1000), SQSTM1/p62 (Abcam, Cambridge, MA, USA, 1:1000), β‐actin (Abcam 1:1000), ASC (Enzo life science, Farmingdale, NY, USA 1:1000), caspase‐1 (Enzo life science, 1/1000), NFĸB (Origene, Rockville, MD, USA, 1:500), GAPDH (GeneTex, Irvine, CA, USA, 1:2000), Atg16L1 (Bethyl, Montgomery, TX, USA, 1:2000), cathepsin D (R&D systems, 1:1000), IL‐1β/IL‐1F2 (R&D systems, 1:1000)] overnight at 4 °C, followed by three 15‐min washes, and then was incubated in blocking buffer containing 0.1% Tween‐20 (Sigma), 0.01% SDS, and IRDye®, 680RD secondary antibody 1:10 000 (Li‐CORE Bioscience, Lincoln, NE, USA) for 1 h at RT. The blot was imaged using the LI‐COR Odyssey imaging system (Li‐CORE Bioscience, Lincoln, NE, USA) and quantified using imageJ software.

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Manufacturer protocol

Download the product protocol from Cell Signaling Technology for LC3A/B (D3U4C) XP® Rabbit mAb #12741 below.

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