Anti-LC3B antibody produced in rabbit

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	1:750 dilution of Primary Antibody

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1:750 dilution of Primary Antibody

Publication protocol

In vitro autophagy activity was determined by the flux assay for LC3B using chloroquine (50 μM) for transient autophagosome-lysosome disruption as previously described [21], [44]. LC3B II and I forms were distinguished by relative molecular weight after separating proteins on a 15% agarose gel. Cells were washed with cold PBS and lysed using RIPA buffer (Sigma-Aldrich; #R0278) containing protease inhibitors (Sigma-Aldrich; #P8340). Protein-containing supernatants were isolated by centrifugation, denatured at 100 °C for 5 min, then briefly disrupted using a water bath sonicator (Branson 5510 model) in four 30-second bursts, and mixed with Laemmli buffer containing 2-mercaptoethanol. Autophagy marker LC3B II levels were assayed by immunoblot using LC3B antibody (1:750 dilution; Sigma-Aldrich; #L7543) on 0.45 µm polyvinylidene fluoride (PVDF) membranes (Immobolin FL; Sigma-Aldrich; IPFL00010). Horseradish peroxidase conjugated goat anti- mouse or rabbit antibodies, respectively, were then used to image primary antibodies with densitometry values normalized to actin. To determine the relevance of STAT6 signaling in autophagy induction, hTEC were lysed in buffer containing NP-40 detergent (0.5%) with 50 mM HEPES (pH 7.0), 5 mM EDTA, 50 mM NaCl, 10 mM Na Pyrophosphate, 50 mM Na fluoride, protease inhibitors (Roche, #11697498001), 100 μM sodium orthovanadate, and freshly prepared phenylmethanesulfonylfluoride (1 mM) dissolved in 200 proof isopropanol. Lysates were centrifuged at 9800g 4 °C. Supernatants were then collected and run on 6–15% gradient acrylamide gel and transferred on PVDF membranes. Proteins were blocked in 5% bovine serum albumin for 1 h and then incubated with primary antibodies to STAT6 (1:500 dilution; Cell Signaling; #D3H4) or phosphorylated STAT6 (Tyr641; 1:500 dilution; Cell Signaling; #C11A12) overnight at 4 °C. STAT6 proteins were detected by chemiluminescence as described above.

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