After twice washing with PBS, the piPSCs (cultured for 5 days) were fixed in 4% paraformaldehyde (pH 7.4) at room temperature for 15 min. We used 0.1% Triton-100 to perforate the membranes at room temperature for 10 min. Then, 10% FBS was used to block the membranes at room temperature for 1 h. The membranes were incubated with primary antibodies, including LIN28A (1∶200; Santa Cruz Biotechnology, USA) and FLAG (1∶1 000; Sigma-Aldrich, USA), for 12 h at 4 °C and then washed three times with PBS. The membranes were then incubated with goat anti-mouse IgG (H+L) secondary antibody Alexa Fluor 488 conjugate (1∶500; ZSGB-BIO, China) at room temperature for 1 h and nuclei were stained with Hoechst33342 (1∶1 000) at room temperature for 5 min (Ma et al., 2019 (link); Wei et al., 2021 (link)).
Lin28a
Manufactured by Santa Cruz Biotechnology
Sourced in United States
LIN28A is a protein that acts as an RNA-binding protein. It is involved in the regulation of messenger RNA (mRNA) and microRNA (miRNA) metabolism, playing a role in cellular differentiation and development.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugate, by ZSGB-BIO (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
β actin, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Gapdh, by Abcam (1 mentions)
3 protocols using lin28a
1
Immunofluorescence Staining of piPSCs
2
Extraction and Detection of Protein Markers
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Cells were lysed as reported previously.58 (link) Total proteins were extracted from AML cell lines. Equal amount of protein extract (60 μg) was transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h with 5% milk (Sigma-Aldrich) at room temperature, and then incubated with primary antibodies directed toward Lin28A (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p21 (Merck Millipore, Billerica, MA, USA) and β-actin (Sigma-Aldrich), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad). Protein bands were visualized and quantified as described previously.59 (link)
3
Western Blotting of Stem Cell Proteins
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Protein (30 µg) from whole cell extracts was used for western blotting analysis. Antibodies used for Western Blotting are as follows: ZIC2 (1:1000, Abcam, Cambridge, MA, USA), LIN28A (1:1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), GAPDH (1:750, Abcam), POLR3G (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA)
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