LC3B Antibody #2775

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	1:2000 dilution of Primary antibody

Protocol tips
1:2000 dilution of Primary antibody

Publication protocol

Western blot analysis
Cells were lysed in RIPA buffer (Sigma-Aldrich). The concentration of the protein samples was determined by the Bradford method (18). Lysates were denatured at 100°C for 10 min and subsequently cooled on ice. A total of 40 µg protein was separated by 8–15% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (GE Healthcare Life Sciences, Chalfont, UK). The primary antibodies used in the present study were: i) β-actin (cat. no. A1978; Sigma-Aldrich; mouse; monoclonal; 1:5,000 in 5% w/v milk); ii) microtubule associated protein 1 light chain 3 [(LC3; cat. no. 2775; rabbit polyclonal; 1:5,000 in 5% w/v bovine serum albumin (BSA; Sangon Biotech Co., Ltd., Shanghai, China); iii) Atg5 (cat. no. 2630; rabbit polyclonal, 1:2,000 in 5% w/v BSA); iv) phosphorylated janus kinase (p-JNK; T183/Y185; cat. no. 4668; rabbit; polyclonal; 1:1,000 in 5% w/v BSA); v) JNK (cat. no. 9252; rabbit; polyclonal; 1:2,000 in 5% w/v BSA); vi) CAV-1 (cat. no. 3238; rabbit; polyclonal; 1:2,000 in 5% w/v BSA); vii) Akt (cat. no. 9272; rabbit; polyclonal; 1:2,000 in 5% w/v BSA); viii) p-Akt (S473; cat. no. 4060; rabbit; polyclonal; 1:2,000 in 5% w/v BSA); ix) Atg7 (cat. no. 2631; rabbit; polyclonal; 1:2,000 in 5% w/v BSA); and x) Beclin1 (cat. no. 3495; rabbit; polyclonal; 1:2,000 in 5% w/v BSA) (all Cell Signaling Technologies, Inc., Danvers, MA, USA).

Laser scanning confocal microscopy
Cells stably expressing ref fluorescent protein (RFP)-LC3 were cultured in live cell imaging culture dishes (Livefocus, Jiangsu, China; cat. no. C-L-8) and subjected to serum starvation in Hank's balanced salt solution (Thermo Fisher Scientific, Inc.) or lentivirus. Living cells were visualized using a Zeiss LSM510 meta-confocal system (Zeiss AG, Oberkochen, Germany). A total of 200 cells were detected at each condition, and significant differences were analyzed by Student's t–test.

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Manufacturer protocol

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