anti cdkn1a p21  (Proteintech)

Journal: PLOS Genetics

Article Title: METTL3 facilitates the translation of CircSIK2 during chicken myogenesis in an m 6 A dependent manner

doi: 10.1371/journal.pgen.1011934

Figure Lengend Snippet: ( A, B ) The transfection efficiency of pCD25-circSIK2, pCD25-circSIK2-MT( A ), and siRNAs ( B ) for circSIK2 ( n = 4). ( C, D ) CCK-8 assay was performed to assess the effect of circSIK2 overexpression and knockdown on myoblast proliferation ( n = 8). ( E ) The statistical results of cell cycle analysis after overexpression and inhibition of circSIK2 in myoblast ( n = 4). ( F, I ) The proliferation rate ( F ) and EdU staining ( I ) of myoblast cells transfected with pCD25-circSIK2, pCD25-circSIK2-MT, and si1-circSIK2 ( n = 4). ( G, H ) mRNA level of CDKN1A , CDKN1B , Cyclin D2 , and PCNA after overexpression and knockdown of circSIK2 ( n = 4). ( J, K ) The protein expression of CDKN1A, CDKN1B, and Cyclin D2 ( J ) and their relative grey value ( K ) was determined by western blot in myoblast cells ( n = 3), western blot was from an aliquot of the same sample but were run and blotted from different gels (*P < 0.05; **P < 0.01, ***P < 0.001).

Article Snippet: The other primary antibodies used for western blots were as follow: anti-flag antibody (Transgen Biotech, China), anti-CDKN1A antibody (bs0741R, Bioss, China), anti-CDKN1B antibody (bs0742R, Bioss, China), anti-CDKN2B antibody (bs4269R, Bioss, China), anti-Cyclin D2 antibody (AF5410, Affinity Biosciences, USA), anti-IGF1R antibody (bs0680R, Bioss, China), anti-INSR antibody (bs0681R, Bioss, China), anti-AKT1 (#9272, Cell Signaling Technology), anti-phospho-AKT1 (#4040, Cell Signaling Technology), anti-MyoD (ab16148, Abcam), anti-MyHC (B103, Developmental Studies Hybridoma Bank), anti-HNRNPA2B1 (Fab6995, Hunan Fenghui Biotechnology), anti-GAPDH (MB001, Bioworld Technology), anti-Tubulin (MB0009, Bioworld Technology).

Techniques: Transfection, CCK-8 Assay, Over Expression, Knockdown, Cell Cycle Assay, Inhibition, Staining, Expressing, Western Blot

Journal: PLOS Genetics

Article Title: METTL3 facilitates the translation of CircSIK2 during chicken myogenesis in an m 6 A dependent manner

doi: 10.1371/journal.pgen.1011934

Figure Lengend Snippet: ( A ) SIK2-176aa promotes the mRNA level of CDKN1A , CDKN1B , CDKN2B , Cyclin D2 , and PCNA . ( B ) The protein expression of CDKN1A, CDKN1B, CDKN2B, and Cyclin D2 was determined by western blot in myoblast cells. ( C ) The statistical results of cell cycle analysis after overexpression of SIK2-176aa. ( D, R ) The EdU staining ( D ) and proliferation rate ( E ) of myoblast cells transfected with pCD3.1-SIK2-176aa-FLAG. ( F, G ) Myotube area ( F ) and MyHC staining ( G ) after transfected with pCD3.1-SIK2-176aa-FLAG. ( H ) SIK2-176aa promotes the mRNA level of GHR-IGF-AKT pathway genes including GHR , IGF2 , IGF1R , INSR , AKT1 , and the myogenic regulatory factor family member including MyoD , MyoG , MyHC , Myomaker , Myf5 , and Myf6 . ( K ) H-E staining of breast muscle fiber cross section infected with LV5-SIK2-176aa or LV5-Control (upper panel) and the statistical data of muscle fiber diameter and cross-sectional area (lower panel). Lentivirus was injected three times into the pectoral muscles of 1-day-old chicks (n = 10) at days 1, 4, and 7 at a dosage of 1 × 10 8 IU/mL. The pectoral muscles were obtained 14 days after the first injection. ( L ) q-RT PCR detection of the injection efficiency of LV5-SIK2-176aa. ( I-J, M-N ) SIK2-176aa promotes the protein expression of IGF1R, INSR, p-AKT1, MyoD, and MyHC in vivo ( I-J ) and in vitro ( M-N ), western blot was from an aliquot of the same sample but were run and blotted from different gels.

Article Snippet: The other primary antibodies used for western blots were as follow: anti-flag antibody (Transgen Biotech, China), anti-CDKN1A antibody (bs0741R, Bioss, China), anti-CDKN1B antibody (bs0742R, Bioss, China), anti-CDKN2B antibody (bs4269R, Bioss, China), anti-Cyclin D2 antibody (AF5410, Affinity Biosciences, USA), anti-IGF1R antibody (bs0680R, Bioss, China), anti-INSR antibody (bs0681R, Bioss, China), anti-AKT1 (#9272, Cell Signaling Technology), anti-phospho-AKT1 (#4040, Cell Signaling Technology), anti-MyoD (ab16148, Abcam), anti-MyHC (B103, Developmental Studies Hybridoma Bank), anti-HNRNPA2B1 (Fab6995, Hunan Fenghui Biotechnology), anti-GAPDH (MB001, Bioworld Technology), anti-Tubulin (MB0009, Bioworld Technology).

Techniques: Expressing, Western Blot, Cell Cycle Assay, Over Expression, Staining, Transfection, Infection, Control, Injection, Muscles, Reverse Transcription Polymerase Chain Reaction, In Vivo, In Vitro

Journal: Journal of Inflammation Research

Article Title: Fatty Acid Transporter CD36 Promotes Ox-LDL-Induced Senescence of Vascular Endothelial Cells in Preeclampsia

doi: 10.2147/JIR.S538337

Figure Lengend Snippet: PE placentas showed significant senescence-related phenotypes. ( A and B ) Western blot analysis of CDKN2A, CDKN1A, and TP53 protein expression in PE (n=4) and NC groups (n=4) ( A ) and quantitative results (B); unpaired t test. ( C – E ) Detecting protein expression level of inflammatory factors in the PE (n=8) and NC placentas (n=8) by Elisa, including IL-6 (C), CXCL-8 ( D ) and TNF-α (E); unpaired t test. ( G – I ) Maternal plasma IL-6 (F), CXCL-8 ( G ) and TNF-α ( H ) levels in the NC (n=18) and PE group (n=18); unpaired t test. ( I ) Result of GSEA. Three senescence-related gene sets were significantly activated in the PE placenta (P-value < 0.05, FDR q-value < 0.25). The data are expressed as the mean ± SD of three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: After blocking with 5% BSA and washing with TBST, the membrane was incubated overnight with the diluted primary antibody, including CD36 (1:1000, 18836-1-AP, Proteintech, China), ox-LDL (1:3000, PAA527Hu08, Cloud-Clone, China), CDKN2A (1:4000, 10883-1-AP, Proteintech, China), CDKN1A (1:2000, 10355-1-AP, Proteintech, China), TP53 (1:20,000, 10442-1-AP, Proteintech, China).

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Clinical Proteomics