- PA5-114352 - Invitrogen Antibodies PLIN5 antibody

Submitted references Leptin Reduces Plin5 m(6)A Methylation through FTO to Regulate Lipolysis in Piglets.

Wei D, Sun Q, Li Y, Li C, Li X, Sun C
International journal of molecular sciences 2021 Sep 30;22(19)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of OXPAT using a OXPAT polyclonal antibody (Product #PA5-114352). Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody at a dilution of 1:500 was used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1:5,000 dilution was used for 1 hour at room temperature. Positive control: Lane 1: Human liver tissue lysate, Lane 2: Human marrow tissue lysate

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometric analysis of OXPAT using JAR cells and a OXPAT polyclonal antibody (Product #PA5-114352). The cells were fixed, permeabilized and stained with the primary antibody at a dilution of 1:100 (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated goat anti-rabbit IgG Secondary antibody at 1:500 dilution for 30 minutes. Unlabeled sample was used as a control (cells without incubation with primary antibody; black).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Leptin improves the body fat composition and weight of piglets by up-regulating the expression of Plin5. ( A ) The leptin kit was used to detect the level of leptin protein in various tissues of piglets. ( B ) The expression of leptin receptors in various tissues of piglets. The effect of leptin treatment on: ( C ) the average daily gain, ( D ) the intramuscular fat content, ( E ) abdominal fat rate and back fat rate and ( F ) blood lipid level etc. ( G ) The change of piglet's body temperature for 15 days after leptin treatment. ( H , I ) The mRNA and protein expression levels of the Plin family were analyzed by real-time quantitative PCR and western blot. ( J ) Immunohistochemical detection of piglet adipose tissue. ( K ) Western blot analysis of lipolytic protein expression. n = 4 in each group, values are means +- SD. vs. control group, * p < 0.05, ** p < 0.01, *** p < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Leptin promotes lipolysis by up-regulating the expression of Plin5 in pig adipocytes. ( A , B ) Real-time quantitative PCR was used to detect the mRNA expression of Plin5, lipolytic genes and mitochondrial complex-related genes in piglets subcutaneous adipose tissue. n = 4 in each group. ( C ) Oil red O staining was used to observe the number and size of LD in the subcutaneous fat tissue of piglets. ( D ) Real-time quantitative PCR was used to detect Plin5 and lipolytic gene expression in porcine adipocytes in vitro treated with 50 nmol/muL leptin for 24 h. The effect of 50 nmol/muL leptin treatment of porcine adipocytes in vitro on: ( E ) the expression of Plin5 and lipolytic protein and ( F ) the mRNA expression of mitochondrial complex-related genes. ( G , H ) Porcine adipocytes in vitro were treated with 50 nmol/muL leptin for 24 h to detect Plin5 immunofluorescence or BODIPY staining analysis. n = 3 for each group of cell samples, values are means +- SD. vs. control group, * p < 0.05, ** p < 0.01.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 FTO up-regulates Plin5 protein expression by inhibiting the m 6 A methylation of Plin5. The effect of FTO interference or overexpression in vitro on: ( A ) Plin family mRNA expression and ( B ) Plin family protein levels. ( C ) The relative M 6 A levels of pig adipocytes in vitro treated with CL and Bet after overexpression or interference with FTO. ( D ) The total M 6 A level of CL-treated pig adipocytes in vitro after overexpression or interference with FTO. ( E ) Plin family mRNA expression in CL-treated pig adipocytes in vitro after overexpression or interference with FTO. ( F ) The total M 6 A level of Bet-treated pig adipocytes after overexpression or interference with FTO. ( G ) Plin family mRNA expression in Bet-treated pig adipocytes in vitro after overexpression FTO. ( H ) Plin5 protein expression in pig adipocytes in vitro treated with CL and Bet after overexpression FTO. ( I , J ) Plin5 3'UTR end M 6 A site mutation vector construction. n = 4 for each group of cell samples, values are means +- SD. vs. control group, * p < 0.05, ** p < 0.01.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Plin5 regulates lipid metabolism and energy metabolism through LD. ( A ) Luciferase reporter assay was used to analyze the effect of M 6 A mutation at the 3'UTR end of Plin5. ( B ) Relative M 6 A level analysis of wild-type and Plin5 mutant. ( C ) Detection of Plin5 mRNA expression of wild type and Plin5 mutant type. ( D ) Plin5 m 6 A methylation analysis of wild type and Plin5 mutant. ( E ) Plin5 protein expression of wild type and Plin5 mutant type. ( F ) Real-time quantitative PCR was used to detect the efficiency of the Plin5 overexpression vector. ( G ) Porcine adipocytes in vitro were transfected with Plin5 overexpression vector and then subjected to BODIPY fluorescent staining of LD. ( H ) Observation of lipid droplet extraction. ( I , J ) Porcine adipocytes in vitro were transfected with Plin5 over-expression vector to detect lipolysis-related genes mRNA or protein levels. n = 4 for each group of cell samples, values are means +- SD. vs. control group, * p < 0.05, ** p < 0.01, *** p < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Plin5 promotes lipid synthesis and enhances mitochondrial beta-oxidation in a lipotoxic model. ( A , B ) Real-time quantitative PCR was used to detect mitochondrial functional gene and beta-oxidation gene mRNA expression after Plin5 overexpression. ( C ) Under the same treatment as ( A , B ), the protein levels of differentially expressed genes were analyzed by Western blotting. ( D ) The kit detected the ATP content of porcine adipocytes transfected with Plin5 overexpression vector. ( E ) The ratio of NAD + /NADH was measured after Plin5 overexpression in porcine adipocytes in vitro. ( F ) 0.4 mmol/L PAL treated porcine adipocytes in vitro for 48 h to detect the mRNA expression of apoptosis-related genes. ( G ) Porcine adipocytes in vitro were treated with 0.4 mmol/L PAL for 48h to perform Tunel staining. ( H ) The FFA level was detected by the kit after treatment of 0.4 mmol/L PAL with porcine adipocytes in vitro for 48 h. ( I ) BODIPY staining after the same treatment in vitro. ( J ) After Plin5 overexpression, 0.4 mmol/L PAL was used to treat porcine adipocytes in vitro for 48 h to detect FFA levels. ( K , L ) After Plin5 was overexpressed, pig adipocytes in vitro were treated with 0.4 mmol/L PAL for 48 h to detect the mRNA levels of lipid synthesis genes and mitochondrial beta-oxidation genes. ( M ) Under the same treatment as ( K , L ), the protein levels of GDAT2 and CPT1a were analyzed by Western blotting. ( N ) The NAD + /NADH ratio is measured after the sam

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