3 × 105 293T cells were transfected using the calcium phosphate method with 2 µg of each TALEN expression plasmid. After two sequential transfections with the hDcr-specific TALEN expression plasmids, individual clones were isolated by serial dilution and assayed by Western Blot for loss of hDcr expression using an antibody that recognizes amino acids 1701–1912 of hDcr (H-212/Santa Cruz). Total cell lysates were transferred to nitrocellulose and following incubation with the primary anti-hDcr antibody, the blot was probed with a secondary α-Rabbit-HRP antibody (A6154/ Sigma-Aldrich) and analyzed as described above. Genomic DNA was isolated (DNeasy/Qiagen) from the parental 293T line and the two NoDice cell lines (2–20 and 4–25). The TALEN-targeted region of hDcr was amplified by PCR and cloned into pcDNA3. Multiple individual clones were sequenced from the parental 293T and NoDice cell lines and three distinct hdcr gene deletions identified in each NoDice cell line.
H 212
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The H-212 is a laboratory equipment designed for cell culture applications. It provides a controlled environment for cell growth and maintenance, with precise regulation of temperature, humidity, and atmospheric composition. The core function of the H-212 is to create and maintain a stable, optimal condition for cell culture processes.
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Lab products found in correlation
Anti sqstm1 p62, by Cell Signaling Technology (1 mentions)
Ecl western blotting substrate, by Promega (1 mentions)
Hrp linked secondary antibody, by Abcam (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Ab51520, by Abcam (1 mentions)
Ab56676, by Abcam (1 mentions)
Ab189073, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti dicer, by Santa Cruz Biotechnology (1 mentions)
Ab2302, by Abcam (1 mentions)
2 protocols using h 212
1
Generation of hDcr Knockout 293T Cell Lines
2
Western Blot Analysis of Autophagy Markers
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Cells were lysed using a lysis buffer (Beyotime, Shanghai, China) and then protein concentration was quantified using a BCA protein assay kit (Beyotime). Then the protein samples were subjected to a conventional western blot analysis. After separation on 10% SDS PAGE gel, the proteins were subsequently transferred onto PVDF membrane and blocked with 5% nonfat milk for 1 h. Membranes were probed overnight at 4°C with primary antibodies: anti‐LC3B (1:3000, ab51520, Abcam, Cambridge, MA, USA), anti‐p62/SQSTM1 (1:1000, #8025, Cell Signaling), anti‐DICER (1:200, H‐212; Santa Cruz Biotechnology), anti‐active caspase‐3 (1:1000, ab2302, Abcam), anti‐tubulin (1:1000, ab56676, Abcam), and anti‐β‐actin (ab189073, 1:1000, Abcam). Band signals were visualized using HRP‐linked secondary antibodies (Abcam) and the ECL Western blotting substrate (Promega, Madison, WI, USA).
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