Plasmids were delivered to HeLa cells with X-tremeGENE HP DNA Transfection Reagent (Roche) according to the manufacturer's instructions, and at a ratio of 2 μl X-tremeGENE HP DNA Transfection Reagent per 1 μg plasmid. Briefly, 2 μg of plasmid and 4 μl of X-tremeGENE HP DNA Transfection Reagent were added per single well of a 12-well plate, and the cells were harvested 36–48 h post-transfection. To test the splicing of cTNT e5, 200 ng of the minigene was delivered to each well. X-tremeGENE HP DNA Transfection Reagent (Mock)- and EGFP-treated HeLa cells were used as controls. For immunoblotting experiments, cells were grown in a 6-well plate and a double amount of plasmid and the transfection reagent were used. Annealed siRNA oligos were delivered to fibroblasts using Lipofectamine 2000 (Invitrogen) per manufacturer's instructions. Oligos targeting human MBNL1 (47 (link)) and MBNL2 (48 (link)) were obtained from Future Synthesis and RiboTask, respectively. The cells were harvested 72 h post siRNA delivery. Lipofectamine (mock)- and AllStars Negative Control siRNA (Qiagen; Ctrl)-treated fibroblasts were used as controls.
Hp dna transfection reagent
Manufactured by Roche
The HP DNA transfection reagent is a laboratory product designed to facilitate the transfer of genetic material, such as DNA, into cells. It provides an efficient method for introducing foreign genetic material into cell lines for various applications, including gene expression studies, protein production, and genetic engineering.
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Lab products found in correlation
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Lipofectamine, by Qiagen (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Eclipse e800, by Nikon (1 mentions)
Mouse monoclonal antibody against actin, by Merck Group (1 mentions)
X tremegene sirna, by Roche (1 mentions)
Vcp sirna, by Thermo Fisher Scientific (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Lenti x concentrator, by Takara Bio (1 mentions)
Lamp1, by Cell Signaling Technology (1 mentions)
Taqman universal pcr master mix, by Roche (1 mentions)
Protein a and g dynabeads, by Thermo Fisher Scientific (1 mentions)
Edem1, by Proteintech (1 mentions)
Disuccinimidyl suberate dss crosslinker, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti β actin, by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
13 protocols using hp dna transfection reagent
1
Plasmid and siRNA Transfection in HeLa and Fibroblasts
2
ATM Mini-Gene Construct Expression
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The 262-bp human ATM mini-gene construct was cloned into the pEGFP-N2 vector. The ATM fragment containing exon 1 with a 72-bp added start codon, 79-bp of intron 1 (containing the 5′-GU-A-AG-3′, splicing recognition site), and 111-bp exon 2, flanked by the engineered EcoRI and BamHI restriction sites, was amplified from the cDNA extracted from the IK-depleted cells. The mini-gene construct (0.5 μg) was transfected with 2 μL of HP DNA transfection reagent (Roche), 24 h after siRNA transfection. After an additional 24 h incubation, protein was extracted using the lysis buffer and immunoblotted with an anti-GFP antibody, and the expression of green fluorescence protein (GFP) was observed by fluorescence microscopy.
3
GFP-LC3 Plasmid Transfection and Fluorescence Microscopy
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According to the protocol provided by Roche, the GFP-LC3 plasmid was transfected into cells using the X-tremeGENE HP DNA Transfection Reagent transfection method. The nuclei were visualized by staining with 4,6-dimidyl-2phenylindole (DAPI), and the green spots of cells were manually counted in five randomly selected areas under a Nikon Eclipse E800 fluorescence microscope.
4
AMFR Protein Expression and Regulation
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Primers, Dynabeads protein A, Superscript VILO cDNA synthesis kit, VCP siRNA, and SVIP siRNA were purchased from Life Technologies (Carlsbad, CA), and X-tremeGENE siRNA and, HP DNA transfection reagent were purchased from Roche Applied Science (Indianapolis, IN). The rabbit polyclonal antibodies against AMFR and ubiquitin and anti-rabbit IgG (conformation specific) were purchased from Cell Signaling (Danvers, MA). The rabbit polyclonal antibody against N-terminus AMFR was purchased from LifeSpan BioScience (Seattle, WA). Polyclonal anti-rabbit antibody against SVIP and mouse monoclonal antibody against actin were purchased from Sigma-Aldrich (St. Louis, MO) Polyclonal anti-rabbit antibodies against p97/VCP and IP lysis buffer were purchased from Thermo Fisher scientific (Waltham, MA). Polyclonal anti-rabbit antibody against AAT was purchased from Dako (Carpentaria, CA).
5
Lentiviral Transduction of XBP1 Isoforms
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Viral coat plasmid (pCMV VSV-G), viral packaging plasmid (pCMV-dR8.2 dvpr) and lentiviral vector (pLenti-C-Myc-DDK) subcloned with open reading frame of either sXBP1 or uXBP1 (Origene) were transfected into 50% confluent 293 T cells using the X-treme gene HP DNA Transfection reagent (Roche). Empty vector was used for control cells. Virus supernatant was collected after 48 and 72 hours, passed through a 0.2 μm filter to remove residual 293 T cells, and stored at 4 °C until use. Viral supernatant was concentrated using Lenti-X concentrator (Clontech), and added to target cells. After 6 hours, media was changed to fresh media. Cells were cultured for 96 hours, and harvested for subsequent analysis.
6
Antibody-Based Detection of ER Stress Proteins
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Rabbit polyclonal antibodies were used to detect ERdj3, EDEM1, and ERp57 (Proteintech, Chicago, IL); others were to detect AAT (DAKO, Carpentaria, CA), GAPDH (Santa Cruz, Dallas, TX), Lamp1, P62, LC3B (all from Cell Signaling, Danvers, MA), and calnexin and calreticulin (both from Stressgen biotechnologies, San Diego, CA). Bafilomycin A1, MG132, Brefeldin A, and mouse monoclonal anti‐β‐actin were purchased from Sigma (St. Louis, MO), and 2C1 against human AAT polymers was purchased from Hycult biotech (Netherlands). Mouse monoclonal antibodies against BiP and AAT were purchased from BD bioscience (San Jose, CA) and R&D systems (Minneapolis, MN), respectively. Alexa Fluor 488 goat anti‐mouse IgG and Alexa Fluor 594 goat anti‐rabbit IgG were purchased from Invitrogen. Primers, Dynabeads protein A and G, Superscript VILO cDNA synthesis kit, and ERdj3 siRNA were purchased from Life Technologies, and HP DNA transfection reagent and TaqMan Universal PCR Master Mix were purchased from Roche Applied Science. Disuccinimidyl suberate (DSS) cross‐linker was purchased from Thermo Fisher scientific (Waltham, MA).
7
Mammalian Cell Culture and Transfection
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293 T cells were maintained in DMEM (Nacalai) containing 10% FBS and 1% penicillin–streptomycin and glutamine (PSG, Invitrogen). CEM and CEM-SS cells were maintained in RPMI1640 containing 10% FBS and 1% PSG. 293 T cells on 6-well plates were transfected with about 1 μg of plasmid DNA in total using X-tremegene HP DNA transfection reagent (Roche) according to manufacturer’s instruction.
8
Transcriptional Regulation via GATA2 and SMAD4
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GATA2 and SMAD4 were separately cloned into the PLVET expression plasmid and the PLVET-GATA2 and PLVET-SMAD4 constructs were established. The SBE4-Luc (16,495) and pGL3-TGFβ1 promoter construct (101,762) were purchased from Addgene. The Renilla control plasmid pGL4.75 (hRluc/CMV) was originally purchased from Promega. For plasmid transfection, V16A cells were reverse transfected with the indicated luciferase reporter plasmids using the X-tremegene HP DNA Transfection Reagent (Roche) according to the manufacturer’s instruction. Forty-eight hours later, Dual-Glo Luciferase Assay System Kit (Promega) was used to examine the luciferase activity of the transfected V16A cells. All data were obtained from five replicates and statistical analysis was performed with a two-tailed student’s test.
9
Dual-Luciferase Reporters Assay for RBM46 Targets
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Dual-luciferase reporters were conducted with Secrete-Pair Dual Luminescence Assay Kit (GeneCopoeia, LF031). The RBM46-bound fragments of targets and their deletion variant, see as Fig. 5C , were inserted into the pEZX-GA02 vector at MCS2 sites. HEK 293 T cells were plated in 12-well plates. Subsequently, a mixture containing 500 ng of reporter plasmid and RBM46 overexpression plasmid was co-transfected to cells with HP DNA Transfection reagent (Roche, 06366546001). And six technical replicates were performed for each group. 72 h later, cells were collected and luciferase activities were assessed according to the manufacturer’s protocols by Microplate luminometer (Berthold, LB960).
10
Luciferase Assay for Atg9B and LAMP1 Genes
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For luciferase reporter experiments, the sequences of 2 kb prior to the first exon of Atg9B and LAMP1 genes were searched in the NCBI database and constructed into the pGL3‐Basic vector (Promega, Madison, WI, USA) by GeneScript (Nanjing, China). Cotransfection of 16E6 plasmid and the reporter gene was performed using Roche HP DNA Transfection Reagent (Roche) following the manufacturer's manual. Briefly, assayed cells were grown to about 60% confluence in a 96‐well tissue culture plate. Cells were cotransfected with pGL3‐Basic promoter and tested plasmids. After 48 hours, the luciferase activities were assessed using the Dual‐Glo Luciferase Assay System (Promega) by measuring the intensity of chemiluminescence in a luminometer (Thermo Fisher Scientific, Waltham, MA, USA). The experiments were performed in triplicate and repeated at least three times with negative controls.
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