Journal: Life

Article Title: Detection of Recombinant Proteins SOX2 and OCT4 Interacting in HEK293T Cells Using Real-Time Quantitative PCR

doi: 10.3390/life13010107

Figure Lengend Snippet: Validation of the interaction of BAP-X and BirA-Y. ( A ) Experimental workflow for the detection of protein–protein interactions between SOX2 and OCT4. ( B ) Western blots of the experiment where X = SOX2 and Y = OCT4. The positions of BAP-fusions and nonspecific signals (NS) are indicated by asterisks. The biotin labeling time was 3 h. The BAP fragment has a His-tag, and the first western blot shows comparable total amounts of BAP-GFP and BAP-SOX2. The Streptavidin blot shows the number of biotin-labeled BAP-fusions which are the result of protein–protein interactions (or proximity) of BAP-SOX2 and BirA-OCT4 in lane 2. A very strong signal was observed in lane 2, and a very weak signal was observed in lane 1, which may be the result of a random collision of BAP-GFP and BirA-OCT4. Treatment with an anti-SOX2 antibody on the third blot indicates recombinant BAP-SOX2 and the absence of a detectable amount of endogenous SOX2. BAP adds a mass shift of 3 kDa, and only a single band was observed on the anti-SOX2 blot in lane 2. ( C ) Western blots of the reciprocal experiment where X = OCT4 and Y = SOX2. The biotin labeling time was 6 h. A comparable expression of BAP-GFP and BAP-OCT4 was detected on anti-His blot lanes 3 and 4. The left blots show the expression, the middle blots show the interaction and the right blots show the absence of the endogenous proteins SOX2 or OCT4. Note that BirA-X (which is actually BirA-8×His-tag-X) fusions were not observed on anti-His blots.

Article Snippet: The vector plasmids pcDNA3-BAP-SOX2 and pOz-humBirA-GFP are available from Addgene (Addgene ID 133281 and 133283, respectively).

Techniques: Western Blot, Labeling, Recombinant, Expressing

Journal: Life

Article Title: Detection of Recombinant Proteins SOX2 and OCT4 Interacting in HEK293T Cells Using Real-Time Quantitative PCR

doi: 10.3390/life13010107

Figure Lengend Snippet: Real-time qRT-PCR amplification of OCT4 and SOX2 in the PUB experiments. Error bars represent the standard deviation.

Article Snippet: The vector plasmids pcDNA3-BAP-SOX2 and pOz-humBirA-GFP are available from Addgene (Addgene ID 133281 and 133283, respectively).

Techniques: Quantitative RT-PCR, Amplification, Standard Deviation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Artemisinin Alleviates Cerebral Ischemia/Reperfusion-Induced Oxidative Damage via Regulating PHB2-Mediated Autophagy in the Human Neuroblastoma SH-SY5Y Cell Line

doi: 10.1155/2022/6568748

Figure Lengend Snippet: SH-SY5Y cells were treated with 3-MA or OGD/R, and the protein expression of LC3II/LC3I was detected by western-blot (a). The quantitative analysis of the protein expression of LC3II/LC3I (b). SH-SY5Y cells were treated with ART for 24 hours under normal conditions after 6h of OGD treatment. The protein expression of TOMM20, PHB2, and LC3II/LC3I was detected by western blot (c). The quantitative analysis of the protein expression of TOMM20, PHB2, and LC3II/LC3I (d-f). Values were expressed as mean ± SD ( n = 3). ∗ P < 0.05 vs. control group, ∗∗ P < 0.01 vs. control group, and ∗∗∗ P < 0.001 vs. control group; # P < 0.05 vs. OGD/R group, ## P < 0.01 vs. OGD/R group, and ### P < 0.001 vs. OGD/R group.

Article Snippet: The primary antibodies used were as follows: Parkin (14060-1-AP, 1 : 1000, Proteintech), prohibitin 2 (12295-1-AP, 1 : 5000, Proteintech), LC3(14600-1-AP, 1 : 1000, Proteintech), p62 (66184-1-lg, 1 : 5000 Proteintech), and β -actin (CW0096M, 1 : 3000, CWBIO).

Techniques: Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Artemisinin Alleviates Cerebral Ischemia/Reperfusion-Induced Oxidative Damage via Regulating PHB2-Mediated Autophagy in the Human Neuroblastoma SH-SY5Y Cell Line

doi: 10.1155/2022/6568748

Figure Lengend Snippet: Silencing PHB2 caused oxidative stress damage (a–d) and leaded to depolarization of MMP (i, j). Silencing PHB2 eliminated the protection of ART against OGD/R-induced oxidative stress in SH-SY5Y cells (e–h) and eliminated the protection of MMP (k, l). Silencing PHB2 eliminated the protective effect of ART on the viability of SH-SY5Y cells (m). Values were expressed as mean ± SD ( n = 6). Bar = 50 μ m. ∗ P < 0.05 vs. control group, ∗∗ P < 0.01 vs. control group, and ∗∗∗ P < 0.001 vs. control group; ## P < 0.01 vs. OGD/R group, ### P < 0.001 vs. OGD/R group; $ P < 0.05 vs. ART+OGD/R group, $$ P < 0.001 vs. ART+OGD/R group, and $$$ P < 0.001 vs. ART+OGD/R group.

Article Snippet: The primary antibodies used were as follows: Parkin (14060-1-AP, 1 : 1000, Proteintech), prohibitin 2 (12295-1-AP, 1 : 5000, Proteintech), LC3(14600-1-AP, 1 : 1000, Proteintech), p62 (66184-1-lg, 1 : 5000 Proteintech), and β -actin (CW0096M, 1 : 3000, CWBIO).

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Artemisinin Alleviates Cerebral Ischemia/Reperfusion-Induced Oxidative Damage via Regulating PHB2-Mediated Autophagy in the Human Neuroblastoma SH-SY5Y Cell Line

doi: 10.1155/2022/6568748

Figure Lengend Snippet: The protein expression of PHB2 and LC3II/LC3I was detected by western blot after silencing PHB2 (a), and the protein expression was quantitatively analyzed (b, c). Cells were treated with ART or OGD/R after silencing PHB2, the protein expression of TOMM20, PHB2, and LC3II/LC3I was detected by western blot (d), and the protein expression of TOMM20, PHB2, and LC3II/LC3I was quantitatively analyzed (e–g). Values were expressed as mean ± SD ( n = 3). ∗ P < 0.05 vs. control group, ∗∗ P < 0.01 vs. control group, and ∗∗∗ P < 0.001 vs. control group; # P < 0.05 vs. OGD/R group, ## P < 0.05 vs. OGD/R group.

Article Snippet: The primary antibodies used were as follows: Parkin (14060-1-AP, 1 : 1000, Proteintech), prohibitin 2 (12295-1-AP, 1 : 5000, Proteintech), LC3(14600-1-AP, 1 : 1000, Proteintech), p62 (66184-1-lg, 1 : 5000 Proteintech), and β -actin (CW0096M, 1 : 3000, CWBIO).

Techniques: Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Artemisinin Alleviates Cerebral Ischemia/Reperfusion-Induced Oxidative Damage via Regulating PHB2-Mediated Autophagy in the Human Neuroblastoma SH-SY5Y Cell Line

doi: 10.1155/2022/6568748

Figure Lengend Snippet: Double immunofluorescence staining was performed to observe the effect of silencing PHB2 on the colocalization expression of PHB2 and LC3. Red fluorescence represents the expression of PHB2 (a–f), green fluorescence represents the expression of LC3 (g–l), and yellow fluorescence represents the colocalization of PHB2 and LC3 (m–r). Bar = 20 μ m. ∗∗ P < 0.01 vs. control group, ∗∗∗ P < 0.001 vs. control group; ## P < 0.01 vs. OGD/R group, ### P < 0.001 vs. OGD/R group; $$ P < 0.01 vs. ART+OGD/R group, $$$ P < 0.001 vs. ART+OGD/R group.

Article Snippet: The primary antibodies used were as follows: Parkin (14060-1-AP, 1 : 1000, Proteintech), prohibitin 2 (12295-1-AP, 1 : 5000, Proteintech), LC3(14600-1-AP, 1 : 1000, Proteintech), p62 (66184-1-lg, 1 : 5000 Proteintech), and β -actin (CW0096M, 1 : 3000, CWBIO).

Techniques: Double Immunofluorescence Staining, Expressing, Fluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Artemisinin Alleviates Cerebral Ischemia/Reperfusion-Induced Oxidative Damage via Regulating PHB2-Mediated Autophagy in the Human Neuroblastoma SH-SY5Y Cell Line

doi: 10.1155/2022/6568748

Figure Lengend Snippet: SH-SY5Y cells stably expressing PHB2-shRNA were treated with ART and CQ for 24 h after OGD treatment, and the LC3II/LC3I ratio was reduced. The protein expression of LC3II/LC3I was detected by western blot (a), and the protein expression wasquantitatively analyzed (n). Values were expressed as mean ± SD ( n = 3). ∗∗ P < 0.01 vs. OGD/R group, ∗∗∗ P < 0.001 vs. OGD/R group; ### P < 0.001 vs. ART+OGD/R group; $$$ P < 0.001 vs. ART+OGD/R +CQ group.

Article Snippet: The primary antibodies used were as follows: Parkin (14060-1-AP, 1 : 1000, Proteintech), prohibitin 2 (12295-1-AP, 1 : 5000, Proteintech), LC3(14600-1-AP, 1 : 1000, Proteintech), p62 (66184-1-lg, 1 : 5000 Proteintech), and β -actin (CW0096M, 1 : 3000, CWBIO).

Techniques: Stable Transfection, Expressing, shRNA, Western Blot