The full-length FAK mRNA sequence was obtained from the NCBI website (NM_153831.3). The different fragments of ANGPTL3 were designed as Fig. 4d . The fragment was obtained by Gene synthesis and cloned into the pcDNA3.1 vector (General Biosystems (Anhui) Co. Ltd.)
Pcdna3.1 vector
Manufactured by General Biosystems
Sourced in China
The PcDNA3.1 vector is a plasmid designed for expression of recombinant proteins in mammalian cell lines. It contains a cytomegalovirus (CMV) promoter, which drives high-level transcription of the inserted gene. The vector also includes a neomycin resistance gene, allowing for selection of transfected cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pcdna3.1 p300, by Addgene (1 mentions)
Pebb flag gcn5, by Addgene (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Lentivirus, by GenePharma (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ham s f 12k, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
5 protocols using pcdna3.1 vector
1
FAK mRNA Sequence and ANGPTL3 Fragment
2
Engineered CXCR4 Constructs for Analysis
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The full-length CXCR4 mRNA sequence was obtained from the NCBI website (NM_003467.2). The wild-type, synonymous mutation (709–726) and NLS mutation (436–447) of CXCR4 sequence were obtained by gene synthesis and cloned into the pcDNA3.1 vector (General Biosystems (Anhui) Co. Ltd.). The detailed mutation is shown in Fig. 2b .
3
Recombinant Protein Expression Evaluation
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The sequences of LAP-TGFB1 (NM_000660.7), HSP90AA1 (NM_005348.4), TIP60 (NM_006388.4), and PCAF (NM_003884.5), as identified in NCBI, were cloned and subjected to mutations. The plasmids pcDNA3β-flag-CBP-HA (Addgene plasmid #32,908), pcDNA3.1-p300 (Addgene plasmid #23,252), and pEBB flag GCN5 (Addgene plasmid #74,784) were kindly provided by Mengshuang Fang. His-LAP-TGFB1, HA-LAP-TGFB1, HA-TGFB1, HA-LAP, HA-LAP-TGFB1 K304Q, HA-LAP-TGFB1 R303A/K304A, flag-TIP60, flag-TIP60 DN, flag-PCAF, HA-CBP, HA-p300, flag-GCN5, and His-HSP90AA1 were subcloned into the pcDNA3.1 (+) vector from General Biosystems, Inc. (Anhui, China). Specific siRNAs and a nontargeting siRNA variant were synthesized by General Biosystems (Supplementary Table 1).
For transfection procedures, both plasmids and siRNAs were introduced into 231 Parental or 231 LuT3 cells using Lipofectamine 3000, adhering strictly to the manufacturer’s protocol. At 48 h post-transfection, cells were analyzed using western blot and real-time qPCR techniques, with a subset reserved for additional experimentation.
For transfection procedures, both plasmids and siRNAs were introduced into 231 Parental or 231 LuT3 cells using Lipofectamine 3000, adhering strictly to the manufacturer’s protocol. At 48 h post-transfection, cells were analyzed using western blot and real-time qPCR techniques, with a subset reserved for additional experimentation.
4
Modulating TPT1-AS1 in Colorectal Cancer
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The CRC cell lines (HCT116, LoVo, SW620 and HT-29) and normal colonic epithelial cell line NCM460 were purchased from American Typer Culture Collection (ATCC) and cultured according to the instructions.
TPT1-AS1 siRNAs specially targeting TPT1-AS1 were designed and synthesized by GenePharma (Shanghai, China). The most interference effectiveness of target sequence was chosen to be packaged lentiviruses by GenePharma (Shanghai, China). A scrambled was used as negative control. To construct TPT1-AS1 downregulated cells model, HCT116 or LoVo (1×105 cells) were mixed with polybrene (5 μg/ml), and Lv-sh-TPT1-AS1(4×108 TU/ml, 5 μL), or Lv-con(2×108 TU/ml, 10 μL). 48 hours later, transfected cells were selected by 20 μg/ml puromycin (Thermo Fisher, USA).
For overexpressional plasmids construction, the TPT1-AS1 sequence and TPT1 cDNA ORF were synthesized and subcloned into the pcDNA3.1 vector (General Biosystems, Chuzhou, China). Plasmids were transfected into cells using Lipofectamine 2000 (Thermo Fisher, USA) following the manufacturers’ instructions. Cells were harvested at 48 h after transfection.
TPT1-AS1 siRNAs specially targeting TPT1-AS1 were designed and synthesized by GenePharma (Shanghai, China). The most interference effectiveness of target sequence was chosen to be packaged lentiviruses by GenePharma (Shanghai, China). A scrambled was used as negative control. To construct TPT1-AS1 downregulated cells model, HCT116 or LoVo (1×105 cells) were mixed with polybrene (5 μg/ml), and Lv-sh-TPT1-AS1(4×108 TU/ml, 5 μL), or Lv-con(2×108 TU/ml, 10 μL). 48 hours later, transfected cells were selected by 20 μg/ml puromycin (Thermo Fisher, USA).
For overexpressional plasmids construction, the TPT1-AS1 sequence and TPT1 cDNA ORF were synthesized and subcloned into the pcDNA3.1 vector (General Biosystems, Chuzhou, China). Plasmids were transfected into cells using Lipofectamine 2000 (Thermo Fisher, USA) following the manufacturers’ instructions. Cells were harvested at 48 h after transfection.
5
Rat Prolactinoma Cell Line Transfection
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We purchased rat prolactinoma MMQ cell lines (ATCC CRL-10609) and GH3 cell lines (ATCC CCL-82.1) from the American Type Culture Collection (ATCC). All cells were grown in Ham's F-12K (Kaighn's) medium (Gibco, Life Technologies), supplemented with 10% fetal bovine serum (Gibco) and 100 U/mL penicillin/ streptomycin (Gibco). We cultured cells in a 5% CO 2 atmosphere at 37°C.
In addition, siRNA of Becn1, miRNA-93-5p inhibitor, miRNA-93-5p mimic and corresponding negative control were purchased from GenePharma (Shanghai, China) (Table 1). Hereinafter, miRNA-93 is specifically referred to as miR-93-5p in the 'Materials and methods' and 'Results' sections. The coding domain sequence of the rat Flag-Atg7 mRNA was amplified by PCR using the rat cDNA as a template and inserted into the pcDNA 3.1 vector (General Biosystems, Inc.). We performed transfection using the Lipofectamine2000 Transfection Reagent (Invitrogen) as per the manufacturer's instructions.
In addition, siRNA of Becn1, miRNA-93-5p inhibitor, miRNA-93-5p mimic and corresponding negative control were purchased from GenePharma (Shanghai, China) (Table 1). Hereinafter, miRNA-93 is specifically referred to as miR-93-5p in the 'Materials and methods' and 'Results' sections. The coding domain sequence of the rat Flag-Atg7 mRNA was amplified by PCR using the rat cDNA as a template and inserted into the pcDNA 3.1 vector (General Biosystems, Inc.). We performed transfection using the Lipofectamine2000 Transfection Reagent (Invitrogen) as per the manufacturer's instructions.
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