tris acetate sa hrp secondary ir 3020 cell signaling technology  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Igf2 regulates early postnatal DPP4 + preadipocyte pool expansion

doi: 10.1101/2025.02.08.637214

Figure Lengend Snippet: ( a ) Projection of two age groups (pre-wean and adult) on isolated scWAT SVCs shown as UMAP plot. ( b ) Cell Clustering of (a) based on cell types. Violin plot of ( c ) preadipocyte markers and ( d ) Igf2 . Adult primary scWAT preadipocytes were serum starved 3h and stimulated with 10 nM IGF2 for 10 min. ( e ) Western blot of phosphorylated (P)/ total (T) AKT and ERK (n=3). Immunoprecipitation (IP) of ( f ) IGF1R (n=2) and ( g ) InsR (n=2). Immunoblotting of P-Tyrosine (TYR) was used to assess the phosphorylation of the receptors after IGF2 stimulation.

Article Snippet: The membrane was blocked with 5% BSA and incubated with primary antibodies overnight at 4°C: phospho-Tyrosine (1:1000, Cell Signaling, 8954), InsR (1:1000, Cell Signaling, 3020), IGF1R (1:1000, Cell Signaling, 9750), B-Actin HRP (only 30 min at RT, 1:5000, Santa Cruz, Sc-47778).

Techniques: Isolation, Western Blot, Immunoprecipitation, Phospho-proteomics

Journal: bioRxiv

Article Title: Igf2 regulates early postnatal DPP4 + preadipocyte pool expansion

doi: 10.1101/2025.02.08.637214

Figure Lengend Snippet: Cultured adult scWAT preadipocytes (Pre) were differentiated with (Dif. Adip + 10nM IGF2) or without (Dif. Adip) 10 nM IGF2. ( a ) mRNA expression of Igf2 (n=5). ( b ) Western blot of InsR, IGF1R and PPARγ. B-Actin was used as loading control (n=5). ( c ) Lipid quantification by Oil red O (ORO) absorbance at 500 nm normalized to DAPI of only differentiated primary adult scWAT preadipocytes (n=5). ( d ) Western blot of phosphorylated (P) and total (T) AKT of protein isolated from cultured pre-wean scWAT preadipocytes that were serum starved for 12 h with 1µg/ml IgG or IGF2 neutralizing antibody (IGF2 Ab) (n=1). ( e ) mRNA levels of Igf2 from differentiated pre-wean scWAT primary preadipocytes that were treated with 1 µg/ml IgG (Dif. Adip + IgG Cnt) or IGF2 neutralizing antibody (Dif. Adip IGF2 Ab) (n=4). ( f ) 100 nM insulin was substituted with either 10 nM IGF2 or insulin during differentiation. mRNA levels of Igf2 and AdipQ (n=3). Data shown by mean ± SEM.

Article Snippet: The membrane was blocked with 5% BSA and incubated with primary antibodies overnight at 4°C: phospho-Tyrosine (1:1000, Cell Signaling, 8954), InsR (1:1000, Cell Signaling, 3020), IGF1R (1:1000, Cell Signaling, 9750), B-Actin HRP (only 30 min at RT, 1:5000, Santa Cruz, Sc-47778).

Techniques: Cell Culture, Expressing, Western Blot, Control, Isolation

Journal: bioRxiv

Article Title: Igf2 regulates early postnatal DPP4 + preadipocyte pool expansion

doi: 10.1101/2025.02.08.637214

Figure Lengend Snippet: Circulating levels of ( a ) insulin (n=8 pre-wean and n=5 adult) and ( b ) IGF2 (n=6 pre-wean and n=5 adult) in pre-wean and adult mice. ( c ) Secreted IGF2 levels in cell culture supernatant of pre-wean and adult primary scWAT preadipocytes (n= 3 pre-wean and n=4 adult). Primary adult preadipocytes were supplemented with (Dif. Adip + 10 nM IGF2) or without (Dif. Adip) 10 nM IGF2 during differentiation. mRNA expression levels of ( d ) Igf2 (n=5) and ( e ) adipogenic markers (n=5). ( f ) Western blot of differentiated adipocytes for InsR, IGF1R, PPARγ. B-Actin was used as loading control (n=5). ( g ) Lipid quantification by Oil Red O (ORO) (n=5). ( h ) Immunocytochemistry of F-Actin (grey) and Lipids (green) (n=4). Primary pre-wean preadipocytes were treated with 1 µg/ml IgG (Dif. Adip + IgG Cnt) or IGF2 neutralizing antibody (Dif. Adip IGF2 Ab) during differentiation. MRNA expression levels of ( i ) Igf2 (n=4) and ( j ) adipogenic markers (n=4). Data shown by mean ± SEM.

Article Snippet: The membrane was blocked with 5% BSA and incubated with primary antibodies overnight at 4°C: phospho-Tyrosine (1:1000, Cell Signaling, 8954), InsR (1:1000, Cell Signaling, 3020), IGF1R (1:1000, Cell Signaling, 9750), B-Actin HRP (only 30 min at RT, 1:5000, Santa Cruz, Sc-47778).

Techniques: Cell Culture, Expressing, Western Blot, Control, Immunocytochemistry

Journal: bioRxiv

Article Title: Igf2 regulates early postnatal DPP4 + preadipocyte pool expansion

doi: 10.1101/2025.02.08.637214

Figure Lengend Snippet: ( a ) Projection of two age groups (pre-wean and adult) on isolated scWAT SVCs shown as UMAP plot. ( b ) Cell Clustering of (a) based on cell types. Violin plot of ( c ) preadipocyte markers and ( d ) Igf2 . Adult primary scWAT preadipocytes were serum starved 3h and stimulated with 10 nM IGF2 for 10 min. ( e ) Western blot of phosphorylated (P)/ total (T) AKT and ERK (n=3). Immunoprecipitation (IP) of ( f ) IGF1R (n=2) and ( g ) InsR (n=2). Immunoblotting of P-Tyrosine (TYR) was used to assess the phosphorylation of the receptors after IGF2 stimulation.

Article Snippet: Unspecific binding sites were blocked using 5% bovine serum albumin (BSA) or non-fat dried milk in TBS-T (1 X TBS containing 0.1% Tween-20) for 1h at RT, followed by incubation with primary antibodies overnight at 4°C: AKT (1:1000, Cell Signaling, 4685), phosphor-AKT Ser 473 (1:1000, Cell Signaling, 9271), ERK (1:1000, Cell Signaling, 4695), phosphor-ERK (1:1000, Cell Signaling, 4377), insulin receptor (InsR) (1:1000, Cell Signaling, 3020), IGF1 receptor (IGF1R) (1:1000, Cell Signaling, 9750), PPARγ (1:1000, Cell Signaling, 2443), B-Actin HRP (only 30 min at RT, 1:5000, Santa Cruz, Sc-47778).

Techniques: Isolation, Western Blot, Immunoprecipitation, Phospho-proteomics

Journal: bioRxiv

Article Title: Igf2 regulates early postnatal DPP4 + preadipocyte pool expansion

doi: 10.1101/2025.02.08.637214

Figure Lengend Snippet: Cultured adult scWAT preadipocytes (Pre) were differentiated with (Dif. Adip + 10nM IGF2) or without (Dif. Adip) 10 nM IGF2. ( a ) mRNA expression of Igf2 (n=5). ( b ) Western blot of InsR, IGF1R and PPARγ. B-Actin was used as loading control (n=5). ( c ) Lipid quantification by Oil red O (ORO) absorbance at 500 nm normalized to DAPI of only differentiated primary adult scWAT preadipocytes (n=5). ( d ) Western blot of phosphorylated (P) and total (T) AKT of protein isolated from cultured pre-wean scWAT preadipocytes that were serum starved for 12 h with 1µg/ml IgG or IGF2 neutralizing antibody (IGF2 Ab) (n=1). ( e ) mRNA levels of Igf2 from differentiated pre-wean scWAT primary preadipocytes that were treated with 1 µg/ml IgG (Dif. Adip + IgG Cnt) or IGF2 neutralizing antibody (Dif. Adip IGF2 Ab) (n=4). ( f ) 100 nM insulin was substituted with either 10 nM IGF2 or insulin during differentiation. mRNA levels of Igf2 and AdipQ (n=3). Data shown by mean ± SEM.

Article Snippet: Unspecific binding sites were blocked using 5% bovine serum albumin (BSA) or non-fat dried milk in TBS-T (1 X TBS containing 0.1% Tween-20) for 1h at RT, followed by incubation with primary antibodies overnight at 4°C: AKT (1:1000, Cell Signaling, 4685), phosphor-AKT Ser 473 (1:1000, Cell Signaling, 9271), ERK (1:1000, Cell Signaling, 4695), phosphor-ERK (1:1000, Cell Signaling, 4377), insulin receptor (InsR) (1:1000, Cell Signaling, 3020), IGF1 receptor (IGF1R) (1:1000, Cell Signaling, 9750), PPARγ (1:1000, Cell Signaling, 2443), B-Actin HRP (only 30 min at RT, 1:5000, Santa Cruz, Sc-47778).

Techniques: Cell Culture, Expressing, Western Blot, Control, Isolation

Journal: bioRxiv

Article Title: Igf2 regulates early postnatal DPP4 + preadipocyte pool expansion

doi: 10.1101/2025.02.08.637214

Figure Lengend Snippet: Circulating levels of ( a ) insulin (n=8 pre-wean and n=5 adult) and ( b ) IGF2 (n=6 pre-wean and n=5 adult) in pre-wean and adult mice. ( c ) Secreted IGF2 levels in cell culture supernatant of pre-wean and adult primary scWAT preadipocytes (n= 3 pre-wean and n=4 adult). Primary adult preadipocytes were supplemented with (Dif. Adip + 10 nM IGF2) or without (Dif. Adip) 10 nM IGF2 during differentiation. mRNA expression levels of ( d ) Igf2 (n=5) and ( e ) adipogenic markers (n=5). ( f ) Western blot of differentiated adipocytes for InsR, IGF1R, PPARγ. B-Actin was used as loading control (n=5). ( g ) Lipid quantification by Oil Red O (ORO) (n=5). ( h ) Immunocytochemistry of F-Actin (grey) and Lipids (green) (n=4). Primary pre-wean preadipocytes were treated with 1 µg/ml IgG (Dif. Adip + IgG Cnt) or IGF2 neutralizing antibody (Dif. Adip IGF2 Ab) during differentiation. MRNA expression levels of ( i ) Igf2 (n=4) and ( j ) adipogenic markers (n=4). Data shown by mean ± SEM.

Article Snippet: Unspecific binding sites were blocked using 5% bovine serum albumin (BSA) or non-fat dried milk in TBS-T (1 X TBS containing 0.1% Tween-20) for 1h at RT, followed by incubation with primary antibodies overnight at 4°C: AKT (1:1000, Cell Signaling, 4685), phosphor-AKT Ser 473 (1:1000, Cell Signaling, 9271), ERK (1:1000, Cell Signaling, 4695), phosphor-ERK (1:1000, Cell Signaling, 4377), insulin receptor (InsR) (1:1000, Cell Signaling, 3020), IGF1 receptor (IGF1R) (1:1000, Cell Signaling, 9750), PPARγ (1:1000, Cell Signaling, 2443), B-Actin HRP (only 30 min at RT, 1:5000, Santa Cruz, Sc-47778).

Techniques: Cell Culture, Expressing, Western Blot, Control, Immunocytochemistry