Journal: International Journal of Molecular Sciences

Article Title: High Dosage Lithium Treatment Induces DNA Damage and p57 Kip2 Decrease

doi: 10.3390/ijms21031169

Figure Lengend Snippet: Effect of lithium on ROS production, DNA damage, and p57 cellular content. ( A ) Effect of different amounts of LiCl on intracellular ROS level in SH-SY5Y cells. Treatment with 1 mM tert -butyl hydroperoxide (TBH) was used as the positive control. The data shown represent the mean of three independent experiments, and the standard deviation (T bar) is reported. A p -value < 0.05 denotes significant difference. ( B ) SH-SY5Y cells were treated with 25 mM LiCl in the presence or absence of N-acetylcysteine (NAC). CTRL refers to cells incubated with 25 mM NaCl; NAC represents cells exposed to 25 mM NaCl plus 150 µM NAC. The growth rate was evaluated by direct cell counting after 24 h incubation. The plot also shows the intracellular ROS levels determined as in ( A ). Data are the mean of three independent experiments, and the standard deviation (T bar) is reported. A p -value <0.05 denotes a significant difference. ( C ) Western blotting analysis of the variation of the amount of γH2AX (pS139H2AX) and H2AX in response to the treatment of SH-SY5Y cells with different amounts of LiCl for 24 h. CTRL is represented by cells incubated with 25 mM NaCl for 24 hours. ( D ) As in panel ( C ), except that p21 Cip1 (p21) and p53 were analyzed in the cell extracts. Equal loading of proteins was verified by determining actin content. ( E ) As in panel ( C ), except that p27 Kip1 was analyzed. Actin content was determined to verify equal loading of proteins. ( F ) As in panel ( C ), except that p57 levels were analyzed. The ratio between p57 and actin signal intensities is reported. ( G ) Lan-5 cells were treated with 25 mM NaCl (CTRL) or with 25 mM Li for 24 and 48 h, and cell extracts were analyzed for p57 content by immunoblotting. Equal loading of proteins was verified by determining actin content. Films at two different exposure times are shown. ( H ) SH-SY5Y cells were transfected for 8 h with 1 µg p57-pcDNA3.1 expression vector. Then, 25 mM NaCl (CTRL) or 25 mM LiCl were added to the cell media for additional 24 h. Thus, equal amounts of proteins were analyzed for p57 content by immunoblotting. The arrows represent the upper and lower p57 signals. The p57-specific signals were quantified as the ratio of the upper and lower signal over actin signal, respectively. Films at two different exposure times (1 and 5 min) are shown. ( I ) SH-SY5Y cells were treated with 25 mM NaCl (CTRL) or 25 mM LiCl for 24 h. After the treatment, cell extracts were analyzed for p57 by bidimensional immunoblotting using anti-p57 mouse monoclonal Ab. The isoforms at the upper level correspond to phosphoforms with an increasing number of phosphate moieties (isoforms from 3 to 6). The lower level includes the unmodified form (signal 1) and the phosphoform with one phosphorylated residue (signal 2). Additional details are described in Materials and Methods, .

Article Snippet: The following primary antibodies were employed: anti-p57 (mouse monoclonal), anti-actin (rabbit polyclonal), anti-p53 (mouse monoclonal), anti-phosphoSer3 cofilin (mouse monoclonal) and anti-cofilin (rabbit polyclonal), anti-γH2AX (mouse monoclonal), anti-PARP1 (mouse monoclonal) and anti-GSK3α/β (mouse monoclonal), anti-HDAC1 (rabbit polyclonal), all purchased from Santa Cruz Biotechnology; anti-p21 Cip1 (mouse monoclonal) and anti phospho-GSK-3α/β (Ser21/9) (rabbit monoclonal) from Cell Signaling Technology (Leiden, The Netherlands); anti-p27 Kip1 (mouse monoclonal), purchased from BD Transduction Laboratories (Franklin Lakes, NJ, USA).

Techniques: Positive Control, Standard Deviation, Incubation, Cell Counting, Western Blot, Transfection, Expressing, Plasmid Preparation, Residue

Journal: International Journal of Molecular Sciences

Article Title: High Dosage Lithium Treatment Induces DNA Damage and p57 Kip2 Decrease

doi: 10.3390/ijms21031169

Figure Lengend Snippet: Molecular mechanisms of lithium’s effect on p57 content. ( A ) Immunoblotting analysis of nuclear and cytosolic fractions of SH-SY5Y cells treated with 25 mM NaCl (CTRL) or LiCl for 24 h. HDAC1 and LDH were analyzed to confirm the efficiency of cell compartment fractionation and equal protein loading. The ratio between p57 and the loading control signal is also reported. ( B ) SH-SY5Y cells were treated with 25 mM NaCl (CTRL) or 25 mM LiCl for 24 h, and the CDKN1C transcript was evaluated by quantitative RT-PCR. The data shown are the mean of three independent experiments, and the standard deviation is reported. A p -value < 0.05 denotes significant difference. ( C ) SH-SY5Y cells were treated with 25 mM NaCl (CTRL) and with 10 and 25 mM LiCl for 24 h. Cell extracts were analyzed for phosphoserine21-phosphoserine9/GSK3α/β (pSer21/pSer9-GSK3α/β) and β-catenin content by immunoblotting. Equal loading of proteins was verified by determining the LDH content. The ratios between pSer21/pSer9-GSK3α/β and GSK3 α/β and β-catenin and LDH band intensities are also shown. ( D ) Effect of SB216763 treatment on CDKN1C expression. SH-SY5Y cells were treated with 25 µM SB216763 or with the vehicle DMSO (CTRL) for 24 h. CDKN1C expression was evaluated by quantitative RT-PCR. The data shown are the mean of three experiments, and the standard deviation is reported. A p -value < 0.05 denotes a significant difference. ( E ) SH-SY5Y cells were treated with different amounts of SB216763 for 24 h. Cell extracts were analyzed for p57 content by immunoblotting. The ratio between p57 and actin signal intensities is also shown. Further details for the densitometric analysis reported in panels ( C ) and ( E ) are described in Materials and Methods, .

Article Snippet: The following primary antibodies were employed: anti-p57 (mouse monoclonal), anti-actin (rabbit polyclonal), anti-p53 (mouse monoclonal), anti-phosphoSer3 cofilin (mouse monoclonal) and anti-cofilin (rabbit polyclonal), anti-γH2AX (mouse monoclonal), anti-PARP1 (mouse monoclonal) and anti-GSK3α/β (mouse monoclonal), anti-HDAC1 (rabbit polyclonal), all purchased from Santa Cruz Biotechnology; anti-p21 Cip1 (mouse monoclonal) and anti phospho-GSK-3α/β (Ser21/9) (rabbit monoclonal) from Cell Signaling Technology (Leiden, The Netherlands); anti-p27 Kip1 (mouse monoclonal), purchased from BD Transduction Laboratories (Franklin Lakes, NJ, USA).

Techniques: Western Blot, Fractionation, Control, Quantitative RT-PCR, Standard Deviation, Expressing

Journal: International Journal of Molecular Sciences

Article Title: High Dosage Lithium Treatment Induces DNA Damage and p57 Kip2 Decrease

doi: 10.3390/ijms21031169

Figure Lengend Snippet: Importance of the decrease of p57 in lithium-dependent DNA damage. ( A ) SH-SY5Y cells were plated in multiple wells at equal density (50–60% of confluency). After 24 h, an equal number of cells were transfected with 300 ng of a p57-encoding pcDNA3.1 plasmid or with a pcDNA3.1 empty vector; then, 8 h after transfection, each cell population was exposed to 25 mM LiCl or to 25 mM NaCl (CTRL) for 24 h. Cells extracts were analyzed by immunoblotting for specific proteins, as reported (p57 on the left, p53 and p21 in the middle, pSer139/H2AX and total H2AX on the right). Two films at different exposure times (30 s and 5 min) for p57 and for pSer139/H2AX (1 and 5 min) are shown. ( B ) Three different siRNAs directed against CDKN1C (named siRNA A, B, and C) were transfected (at 100 nM concentration) in SH-SY5Y cells (see Materials and Methods, ). Forty-eight hours after transfection, cells were harvested, and total RNA was prepared. CDKN1C expression was evaluated by quantitative RT-PCR. The data shown are the mean of three experiments, and the standard deviation (T bar) is reported. ( C ) SH-SY5Y cells were first transfected with a mixture of anti-p57 siRNA A and siRNA C (siRNA A/C) for 48 h and thereafter exposed to 1 mM and 25 mM LiCl for 24 h; a specific control included cells incubated for 24 h with 25 mM NaCl after 48 h of transfection with a scramble siRNA. The transfected cells were also compared to SH-SY5Y cells treated with the two different Li concentrations (1 mM and 25 mM) for 24 h. In addition, SH-SY5Y cells were treated with 2 µM CPT (camptothecin) for 5 h after transfection (or not) with siRNAs. Then, equal amounts of proteins were analyzed for p57 content by immunoblotting. LDH was used as equal loading control. Three different exposure times are shown to highlight signal differences. ( D ) SH-SY5Y cells were treated with NaCl (CTRL) or with LiCl at two different concentrations in the presence or absence of the siRNA mixture. Moreover, cells were treated with 2 µM CPT. After 24 h, cell extracts were prepared and analyzed. In ( a ), p57 and actin levels were investigated while, in ( b ), γH2AX (pS139H2AX) and H2AX were studied. The ratios between p57/actin and γH2AX/H2AX were also reported. ( E ) The samples in ( D ) were analyzed for phosphoserine3-cofilin and cofilin content. The ratio between phosphocofilin and cofilin signal intensities is also shown. Details of the densitometry analysis are in Materials and Methods, .

Article Snippet: The following primary antibodies were employed: anti-p57 (mouse monoclonal), anti-actin (rabbit polyclonal), anti-p53 (mouse monoclonal), anti-phosphoSer3 cofilin (mouse monoclonal) and anti-cofilin (rabbit polyclonal), anti-γH2AX (mouse monoclonal), anti-PARP1 (mouse monoclonal) and anti-GSK3α/β (mouse monoclonal), anti-HDAC1 (rabbit polyclonal), all purchased from Santa Cruz Biotechnology; anti-p21 Cip1 (mouse monoclonal) and anti phospho-GSK-3α/β (Ser21/9) (rabbit monoclonal) from Cell Signaling Technology (Leiden, The Netherlands); anti-p27 Kip1 (mouse monoclonal), purchased from BD Transduction Laboratories (Franklin Lakes, NJ, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Concentration Assay, Expressing, Quantitative RT-PCR, Standard Deviation, Control, Incubation

Journal: Foods

Article Title: The Influence of Red Cabbage Extract Nanoencapsulated with Brassica Plasma Membrane Vesicles on the Gut Microbiome of Obese Volunteers

doi: 10.3390/foods10051038

Figure Lengend Snippet: Extract composition of the red cabbage aqueous extract for the free and the nanoencapsulated treatment. The numbers show the average values ( n = 3) ± standard error. Different letters in a row indicate statistically significant differences in the HSD Tukey’s test ( p < 0.05).

Article Snippet: The standards employed for quantification were sulforaphane (SFN), SFN-glutathione (SFN-GSH), SFN-cysteine (SFN-CYS), SFN-N, acetylcysteine (SFN-NAC), iberin, and indole-3-carbinol (I3C) from Santa Cruz Biotechnology (Dallas, TX, USA), via Quimigen S.L. (Madrid, Spain).

Techniques:

Journal: Foods

Article Title: The Influence of Red Cabbage Extract Nanoencapsulated with Brassica Plasma Membrane Vesicles on the Gut Microbiome of Obese Volunteers

doi: 10.3390/foods10051038

Figure Lengend Snippet: Percentage of the relative abundance of ( A ) sulforaphane (SFN), ( B ) indole-3-carbinol, and ( C ) iberin, when feeding the dynamic colonic-gastrointestinal digester (D-CGID) with the red cabbage aqueous extract, both free and nanoencapsulated ( n = 3 ± SE). The reference extract was taken as 100% and a two-way ANOVA analysis with an HSD Tukey’s test as a post hoc test was performed. Different letters indicate statistically significant differences ( p < 0.05).

Article Snippet: The standards employed for quantification were sulforaphane (SFN), SFN-glutathione (SFN-GSH), SFN-cysteine (SFN-CYS), SFN-N, acetylcysteine (SFN-NAC), iberin, and indole-3-carbinol (I3C) from Santa Cruz Biotechnology (Dallas, TX, USA), via Quimigen S.L. (Madrid, Spain).

Techniques:

Journal: Cell death discovery

Article Title: HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis.

doi: 10.1038/s41420-024-01808-8

Figure Lengend Snippet: Fig. 1 HOOK3 was decreased in GC tissues and negatively related to poor prognosis in GC patients. A Total of 81 GC samples and 70 normal adjacent tissues were subjected to immunohistochemistry (IHC) analysis to assess the expression of HOOK3. An illustrative image representing the results is provided. B The overall survival of GC patients was stratified based on high and low HOOK3 expression groups, and Kaplan-Meier curves were generated. C Multivariate Cox regression analysis was conducted to evaluate the association between HOOK3 expression and overall survival, as depicted in the forest plots. D Western blotting was employed to measure HOOK3 protein expression in GES-1, AGS, HGC27, MKN28, and MKN45 cells, with GAPDH serving as a loading control. The Image J program was utilized for the quantification of Western blot band densities. The resulting values were presented as means with standard deviations (SD), and statistical significance was evaluated using Student’s t-test. **P < 0.01, and ***P < 0.001.

Article Snippet: The Western blotting procedure employed a selection of primary antibodies, namely HOOK3 (Proteintech, #15457-1-AP), VEGFA (Proteintech, #19003-1-AP), SP1 (Proteintech, #21962-1-AP), and GAPDH (Proteintech, #60004-1-Ig).

Techniques: Immunohistochemistry, Expressing, Generated, Western Blot, Control

Journal: Cell death discovery

Article Title: HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis.

doi: 10.1038/s41420-024-01808-8

Figure Lengend Snippet: Fig. 2 HOOK3 knockdown promoted the proliferation, migration, and invasion in GC cells. A The protein expression of HOOK3 in MKN-28 and AGS cells was examined following transfection with si-HOOK3-1 or si-HOOK3-2. GAPDH was used as a loading control. The densities of Western blot bands were quantified using the ImageJ program. B The proliferation of AGS and MKN-28 cells was assessed after transfection with si-HOOK3-1 or si-HOOK3-2 using a CCK-8 assay. C Colony formation assays were performed on AGS and MKN-28 cells transfected with si- HOOK3-1 or si-HOOK3-2. D EdU analysis was conducted to evaluate the proliferative ability of AGS and MKN-28 cells treated with si-HOOK3-1 or si-HOOK3-2. Representative images were provided, and a scale bar of 100 μm was included. The bar graph shows the statistical analysis of the percentage of EdU-positive cells in transfected GC cells. E Transwell migration and invasion assays were conducted on AGS and MKN-28 cells transfected with si-HOOK3-1 or si-HOOK3-2. F Apoptosis assays were conducted were performed on AGS and MKN-28 cells transfected with si-HOOK3-1 or si-HOOK3-2. The experiments were performed in triplicate. The data are presented as the mean with standard deviation (SD), and statistical significance was determined using Student’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The Western blotting procedure employed a selection of primary antibodies, namely HOOK3 (Proteintech, #15457-1-AP), VEGFA (Proteintech, #19003-1-AP), SP1 (Proteintech, #21962-1-AP), and GAPDH (Proteintech, #60004-1-Ig).

Techniques: Knockdown, Migration, Expressing, Transfection, Control, Western Blot, CCK-8 Assay, Standard Deviation

Journal: Cell death discovery

Article Title: HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis.

doi: 10.1038/s41420-024-01808-8

Figure Lengend Snippet: Fig. 3 HOOK3 overexpression inhibited the proliferation, migration, and invasion in GC cells. A The protein expression of HOOK3 was examined in MKN-28 and HGC-27 cells with stable overexpression of HOOK3. GAPDH was used as a loading control. The quantification of band densities in Western blot analysis was performed using the ImageJ program. B The proliferation of MKN-28 and HGC-27 cells with stable overexpression of HOOK3 was assessed using a CCK-8 assay. C The colony formation assay was conducted to evaluate the ability of HOOK3 overexpressing MKN-28 and HGC-27 cells to form colonies. D EdU analysis was carried out to measure the proliferative ability of MKN-28 and HGC-27 cells with HOOK3 overexpression. Representative images are presented, with a scale bar of 100 μm. The percentage of EdU-positive cells in transfected GC cells was statistically analyzed and shown in the bar graph. E Transwell migration and invasion assays were performed to examine the migratory and invasive capacities of MKN-28 and HGC-27 cells with HOOK3 overexpression. F Apoptosis assays were performed on MKN-28 and HGC-27 cells with HOOK3 overexpression. The experiments were conducted in triplicate. The data were represented as means with standard deviation (SD), and statistical significance was assessed using Student’s t-test. **P < 0.01, and ***P < 0.001.

Article Snippet: The Western blotting procedure employed a selection of primary antibodies, namely HOOK3 (Proteintech, #15457-1-AP), VEGFA (Proteintech, #19003-1-AP), SP1 (Proteintech, #21962-1-AP), and GAPDH (Proteintech, #60004-1-Ig).

Techniques: Over Expression, Migration, Expressing, Control, Western Blot, CCK-8 Assay, Colony Assay, Transfection, Standard Deviation

Journal: Cell death discovery

Article Title: HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis.

doi: 10.1038/s41420-024-01808-8

Figure Lengend Snippet: Fig. 4 VEGFA was involved in HOOK3-mediated proliferation, migration, and invasion in GC cells. A Volcano plot of differentially expressed genes (DEGs) in MKN-28 cells and HOOK3-knockdown MKN-28 cells. B Gene Ontology (GO) enrichment analysis of the top 0.5% negatively correlated genes among the DEGs. C The frequency of genes related to migration and invasion observed in the GO enrichment analysis. D The protein levels of VEGFA were measured in HOOK3-overexpressing MKN-28 and HGC-27 cells, as well as HOOK3-knockdown MKN-28 and HGC-27 cells, with GAPDH serving as a loading control. E The protein levels of VEGFA were assessed in HOOK3-overexpressing MKN-28 and HGC-27 cells treated with a plasmid that overexpresses VEGFA, with GAPDH used as a loading control. F The proliferation of MKN- 28 and HGC-27 cells overexpressing HOOK3 and treated with a plasmid overexpressing VEGFA was evaluated using a CCK-8 assay. G Colony formation assays were performed on MKN-28 and HGC-27 cells with HOOK3 overexpression and treated with a VEGFA-overexpressing plasmid. H EdU analysis was conducted on MKN-28 and HGC-27 cells overexpressing HOOK3 after treatment with a plasmid that overexpresses VEGFA. I Transwell migration and invasion assays were carried out on HOOK3-overexpressing MKN-28 and HGC-27 cells treated with a VEGFA-overexpressing plasmid. The experiments were conducted in triplicate. The values were represented as means with standard deviation (SD), and statistical significance was determined using Student’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The Western blotting procedure employed a selection of primary antibodies, namely HOOK3 (Proteintech, #15457-1-AP), VEGFA (Proteintech, #19003-1-AP), SP1 (Proteintech, #21962-1-AP), and GAPDH (Proteintech, #60004-1-Ig).

Techniques: Migration, Knockdown, Control, Plasmid Preparation, CCK-8 Assay, Over Expression, Standard Deviation

Journal: Cell death discovery

Article Title: HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis.

doi: 10.1038/s41420-024-01808-8

Figure Lengend Snippet: Fig. 5 HOOK3 inhibited VEGFA expression via SP1. A The intersection of transcription factors predicted by three databases, GTRD (red circle), JASPAR (green circle), and TFDB (blue circle), was analyzed. B The expression of YY1, SP1, and ZEB1 was analyzed by RT-qPCR in HOOK3 knockdown MKN-28 and HGC-27 cells. C The expression of YY1, SP1, and ZEB1 was analyzed by RT-qPCR in HOOK3-overexpressing MKN-28 and HGC-27 cells. D Western blot analysis was performed to evaluate the expression of SP1 in either HOOK3 knockdown or HOOK3- overexpressing MKN-28 cells. GAPDH was used as the control. The band densities from Western blot were quantified using the ImageJ program. E Western blot analysis was performed to evaluate the expression of SP1 in either HOOK3-knockdown or HOOK3-overexpressing HGC-27 cells. GAPDH was used as the control. The band densities from Western blot were quantified using the ImageJ program. F A schematic diagram illustrating the presence of three SP1 binding sites (P1: −367nt to −358nt, P2: −642nt to −632nt, and P3: −76nt to −68nt) in the 5′ region of the VEGFA promoter. G ChIP analysis was conducted to assess the binding of SP1 to the VEGFA promoter in MKN-28 cells. Normal rabbit IgG was used as the control. H The luciferase activity, responsive to SP1, was measured in MKN-28 and HGC-27 cells transfected with HOOK3 overexpressing plasmid and SP1 overexpressing plasmid. I Western blot analysis was performed to evaluate the expression of VEGFA and SP1 in HOOK3-overexpressing MKN-28 cells transfected with SP1 overexpression plasmid. GAPDH was used as the control. The band densities from Western blot were quantified using the ImageJ program. J Western blot analysis was performed to evaluate the expression of VEGFA and SP1 in HOOK3-overexpressing HGC-27 cells transfected with SP1 overexpression plasmid. GAPDH was used as the control. The band densities from Western blot were quantified using the ImageJ program. The experiments were conducted in triplicate. The mean values with standard deviation (SD) were presented, and the statistical significance was determined using Student’s t-test. Nonsignificant results were denoted as “ns”, while significance levels were shown as *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The Western blotting procedure employed a selection of primary antibodies, namely HOOK3 (Proteintech, #15457-1-AP), VEGFA (Proteintech, #19003-1-AP), SP1 (Proteintech, #21962-1-AP), and GAPDH (Proteintech, #60004-1-Ig).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Western Blot, Control, Binding Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Over Expression, Standard Deviation

Journal: Cell death discovery

Article Title: HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis.

doi: 10.1038/s41420-024-01808-8

Figure Lengend Snippet: Fig. 6 HOOK3 overexpression suppressed proliferation and metastasis of GC cells in vivo. A The image displayed is representative of HOOK3-overexpressing MKN28 tumors grown in nude mice. The volumes of these tumors were evaluated in groups containing four mice each. B Representative images of immunohistochemistry (IHC) staining for Ki-67 and VEGFA and TUNEL staining were captured in tumor tissues from the OE-HOOK3 and OE-NC groups. The scale bar represents 50 μm. C A murine lung metastasis model was established using HOOK3-overexpressing GC cells and control GC cells, with three mice in each group. D Representative images of hematoxylin and eosin (HE) stains were obtained from lung metastasis in the OE-HOOK3 and OE-NC groups. The scale bar represents 50 μm. The mean values along with standard deviations (SD) were used to represent the data, and statistical significance was determined using Student’s t-test. **P < 0.01.

Article Snippet: The Western blotting procedure employed a selection of primary antibodies, namely HOOK3 (Proteintech, #15457-1-AP), VEGFA (Proteintech, #19003-1-AP), SP1 (Proteintech, #21962-1-AP), and GAPDH (Proteintech, #60004-1-Ig).

Techniques: Over Expression, In Vivo, Immunohistochemistry, TUNEL Assay, Staining, Control

Journal: Cell death discovery

Article Title: HOOK3 suppresses proliferation and metastasis in gastric cancer via the SP1/VEGFA axis.

doi: 10.1038/s41420-024-01808-8

Figure Lengend Snippet: Fig. 7 The mechanism of HOOK3 regulating VEGFA/SP1 in GC. Schematic diagram of regulation mechanism of HOOK3/VEGFA/SP1 axis in GC.

Article Snippet: The Western blotting procedure employed a selection of primary antibodies, namely HOOK3 (Proteintech, #15457-1-AP), VEGFA (Proteintech, #19003-1-AP), SP1 (Proteintech, #21962-1-AP), and GAPDH (Proteintech, #60004-1-Ig).

Techniques: