rabbit anti-p-src y418  (Thermo Fisher)

Journal: Mediators of Inflammation

Article Title: Toll-Like Receptor 4 Mediated Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation in Vascular Smooth Muscle Cells via Src and Sirt1/3 Pathway

doi: 10.1155/2021/6639252

Figure Lengend Snippet: oxLDL regulated expression of toll-like receptor family, ROS-related genes, and sirtuin family as well as activation of Src. After oxLDL stimulation, the mRNA or protein expression of genes was detected by real-time PCR or western blot in VSMCs. The phosphorylation of Src was detected using western blot. The untreated group served as control (Con), and β -actin served as an internal reference gene to normalize protein expression. All of the real-time PCR results were calculated by 2 - ΔΔ CT method; the western blot results were calculated using grayscale value. (a) The mRNA expression of the toll-like receptor family ranging from Tlr1 to Tlr13 . ( n = 3 per group, results were expressed as mean ± SD; ∗ P < 0.01, as compared with the Con group.) (b) Upper: the phosphorylation of Src (Tyr-418, Y418) was detected after 1-hour stimulation by different doses (12.5, 25, and 50 μ g) of oxLDL in VSMCs. Bottom: the protein expression of ROS generation (Nox2 and Nox4) and elimination-related (Mnsod) genes in VSMCs, after stimulating by different doses (12.5, 25, and 50 μ g) of oxLDL for 48 hours. (c) After incubation with different doses (12.5, 25, and 50 μ g) of oxLDL, the protein levels of the sirtuin family (Sirt1-7) were measured. ( n = 3 per group, results were expressed as mean ± SD; ∗∗ P < 0.01 as compared with the Con group).

Article Snippet: Additionally, primary antibodies of β -actin (Cat#3700), p-Src Y418 (Cat#6943), t-Src (Cat#2123), Mnsod (Cat#13141), Sirt1 (Cat#2314), Sirt2 (Cat#12650), Sirt3 (Cat#5490), Sirt5 (Cat#8782), Sirt6 (Cat#12486), Sirt7 (Cat#5360), and tlr4 (Cat#14358) as well as the second antibody-conjunction HRP anti-mouse/rabbit (Cat#7076/Cat#7074) were gained from CST (MA, USA).

Techniques: Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Incubation

Journal: Mediators of Inflammation

Article Title: Toll-Like Receptor 4 Mediated Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation in Vascular Smooth Muscle Cells via Src and Sirt1/3 Pathway

doi: 10.1155/2021/6639252

Figure Lengend Snippet: Knockdown or inactivation of Src regulated lipid accumulation and cellular phenotype in VSMCs. (a, b) Tlr4(-) or NC were treated with or without oxLDL (50 μ g/mL) for 1 hour; the activation of Src (Tyr-418, Y418) had been detected by western blot. ( n = 3 per group, results were expressed as mean ± SD; ∗∗ P < 0.01, as compared with oxLDL-untreated NC group; ## P < 0.01, as compared with oxLDL-treated NC group.) Src was knockdown by siRNA transfection within VSMCs. The Src-specific antagonist, PP2, was used to block Src activation, with PP3 serving as the negative control. The VSMCs were treated with and without 50 μ g/mL oxLDL for 48 hours. (c, d) The Src-specific [Src (-)] and negative control (NC) siRNA were, respectively, transfected into VSMCs for 72 hours, and the knockdown efficiency was detected. ( n = 3 per group, results were expressed as mean ± SD; ∗∗ P < 0.01, as compared with the NC group.) (e) Nile Red (orange) was used to label the lipid, and the concentration of intracellular lipid was measured. ( n = 5 per group, results were expressed as mean ± SD; ∗∗ P < 0.01, as compared with the untreated group; @@ P < 0.01, the oxLDL-treated Src siRNA group compared with the oxLDL-treated NC group; ## P < 0.01, the oxLDL-treated PP2 group compared with the oxLDL-treated PP3 group.) (f, g) Western blot was conducted to measure the VSMC contractile phenotype markers (Myh11 and α Sma) and foam cell markers (Mac2 and Cd68). All the western blot results were calculated using grayscale value, and β -actin served as an internal reference gene to normalize protein expression. ( n = 3 per group, results were expressed as mean ± SD; ∗∗ P < 0.01, as compared with the control group (untreated group); @@ P < 0.01, the oxLDL-treated Src siRNA group compared with the oxLDL-treated NC group; ## P < 0.01, the oxLDL-treated PP2 group compared with oxLDL-treated PP3 group).

Article Snippet: Additionally, primary antibodies of β -actin (Cat#3700), p-Src Y418 (Cat#6943), t-Src (Cat#2123), Mnsod (Cat#13141), Sirt1 (Cat#2314), Sirt2 (Cat#12650), Sirt3 (Cat#5490), Sirt5 (Cat#8782), Sirt6 (Cat#12486), Sirt7 (Cat#5360), and tlr4 (Cat#14358) as well as the second antibody-conjunction HRP anti-mouse/rabbit (Cat#7076/Cat#7074) were gained from CST (MA, USA).

Techniques: Activation Assay, Western Blot, Transfection, Blocking Assay, Negative Control, Concentration Assay, Expressing