Lc3ii

Manufactured by Thermo Fisher Scientific

Sourced in United States

LC3II is a lab equipment product for the analysis and quantification of proteins involved in autophagy, a cellular process. It is used to measure the levels of the autophagy marker protein LC3-II, which is associated with the formation of autophagosomes. The LC3II product provides a tool for researchers to study and monitor autophagy in various biological systems.

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Lab products found in correlation

4 protocols using lc3ii

Investigating OPN, TGF-β, and LC3 in HOb Cells

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HOb cells were seeded and 24 h later samples were added. After three days, cells were collected and lysed. Protein concentrations were measured and sodium dodecyl sulfate polyacrylamide gel electrophoresis was done to separate cell lysates. Lysates were transferred to polyvinylidene difluoride membranes, followed by probing with primary antibodies. Mouse anti-OPN and rabbit-anti TGF-β were purchased from GeneTex Inc. and were used at dilutions 1:1000. Rabbit anti-LC3I and LC3II were purchased from Thermo Fisher Scientific and were used at dilutions 1:1000. Rabbit anti-pSmad 1/5/8 were used at the dilutions of 1:500 and were purchased from Santa Cruz Biotechnology (CA, USA). Rabbit anti-β-actin was purchased from Abcam and was used at dilutions of 1:500. Secondary antibodies, goat anti-mouse IgG HRP from Thermo Fisher Scientific at 1:3000 and goat anti-rabbit IgG H&L HRP from Abcam at 1:3000 were used (Tai et al., 2020 ).

Imtiyaz Z., Lin Y.T., Cheong U.H., Jassey A., Liu H.K, & Lee M.H. (2020). Compounds isolated from Euonymus spraguei Hayata induce ossification through multiple pathways. Saudi Journal of Biological Sciences, 27(9), 2227-2237.

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HPDF Autophagy Gene Expression

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Total RNAs of HPDLFs were extracted using a miRNeasy Mini Kit (Qiagen). One microgram of each RNA was transcribed to complementary DNA and TaqMan probes (IL‐1β, Dec2, ATG5, Beclin1, LC3‐I, LC3‐II, and ACTB; Thermo Fisher Scientific) were used.

Oka S., Li X., Sato F., Zhang F., Tewari N., Chen C., Zhong L., Makishima M., Liu Y, & Bhawal U.K. (2020). Dec2 attenuates autophagy in inflamed periodontal tissues. Immunity, Inflammation and Disease, 9(1), 265-273.

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Quantitative Western Blot Analysis

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Western blot analysis was performed as previously described [15 (link)]. The concentration of proteins was determined by using a protein assay kit (Bio-Rad, USA). 40 μg of proteins was electrophoresed on SDS-PAGE gels and then transferred onto polyacrylamide difluoride (PVDF) membrane (Millipore, Massachusetts, USA). Membranes were incubated with respective primary antibodies (cleaved-caspase-3, 1 : 1000, Abcam; Bcl-2, 1 : 1000, Thermo Fisher; mTOR, 1 : 2000, Abcam; beclin-1, 1 : 1000, Thermo Fisher; LC3II, 1 : 1000, Thermo Fisher) overnight at 4°C, followed by incubation with horseradish peroxidase- (HRP-) conjugated goat anti-rabbit (1 : 5000, Thermo Fisher) or goat anti-mouse IgG secondary antibodies (1 : 10000, Thermo Fisher) for 1 hour at room temperature. Protein bands were visualized by an enhanced chemiluminescence system (ECL kit, GE Healthcare, USA). Quantification of band intensity was analyzed using ImageJ software. β-Actin (1 : 5000, Thermo Fisher) served as loading condition.

Du L., Yang Z., Sheng H., Liu M, & Sun Q. (2022). Effects of Long-Term Vagus Nerve Electrical Stimulation Therapy on Acute Cerebral Infarction and Neurological Function Recovery in Post MCAO Mice. Oxidative Medicine and Cellular Longevity, 2022, 8131391.

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Protein Extraction and Western Blot Analysis

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The total proteins of CC cells were extracted by lysis buffer. The extracts were centrifugated at 12,000 ×g for 30 min at 4°C. The protein concentration was then detected by BCA kit. The proteins were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis, followed by transferring to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated in 5% nonfat milk at 25°C for 2 h, and then incubated with primary antibodies (LC3I, LC3II, Beclin1, and p62 were purchased from Thermo Fisher Scientific, Waltham, MA, USA) at 4°C overnight. After washing with tris-buffered saline with Tween-20 (TBST), the membranes were incubated with secondary antibodies for 1 h. The bands were visualized using enhanced chemiluminescence kit (Santa Cruz Biotechnology, CA, USA) and recorded with Tanon 5200 (Tanon, Shanghai, China).

Su G., Chen X, & Yang H. (2022). Pantoprazole promotes the sensitivity of cervical cancer cells to cisplatin by inhibiting cisplatin-induced autophagy. Journal of cancer research and therapeutics, 18(2).

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