Journal: Frontiers in Physiology

Article Title: Acetate suppresses myocardial contraction via the short-chain fatty acid receptor GPR43

doi: 10.3389/fphys.2022.1111156

Figure Lengend Snippet: GLPG0974 prevented the acetate-induced inhibition of myocardial contraction. Photomicrographs showing GPR43 and RyR2 were co-expressed in myocardial cell (A) , GPR43 was labeled red, RyR2 was labeled red green and DAPI was labeled blue in the nucleus; scale bar, 20 μm. The GPR43 protein expression in myocardial cells was observed by Western blot (B) . Representative traces of myocardial contraction in isolated myocardial cells (C) . The sarcomere contraction amplitude (D) , diastolic sarcomere length (E) , maximum contraction velocity (F) , and maximum relaxation velocity (G) in the isolated myocardial cell. N = 5, n = 11. N: the number of rats, n: the number of cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After blocking with 10% normal goat serum (Solarbio, Beijing, China), cells were incubated with GPR43 (dilution 1:100, Cat# AFR-032, RRID: AB_2756592, Alomone labs, Israel) and the type 2 ryanodine receptor (RyR2, dilution 1:100, Cat# ab2827, RRID: AB_2183052, Abcam, United States) antibodies for 12 h in 4°C.

Techniques: Inhibition, Labeling, Expressing, Western Blot, Isolation

Journal: International Journal of Biological Sciences

Article Title: Circ-HIPK3 Strengthens the Effects of Adrenaline in Heart Failure by MiR-17-3p - ADCY6 Axis

doi: 10.7150/ijbs.36149

Figure Lengend Snippet: MiR-17-3p was the downstream target of circ-HIPK3. (A) Results of WB showing that p-RyR2 and p-PLN increased in circ-HIPK3 overexpressed NMCMs and vice versa. (B) QRT-PCR showing that PLN and RyR2 changed little in circ-HIPK3 over- or under- expressed NMCMs. NS = not significant. (C) Schematic image showing that seven possible binding sites of miR-17-3p to circ-HIPK3 were found. (D) Dual-luciferase reporter gene assay showed miR-17-3p could decrease the fluorescence density of NMCMs transfected with pmirGLO- wt -circ-HIPK3. * p<0.05 . (E) FISH assay showed circ-HIPK3 (red) can interact with miR-17-3p (green), which mainly were around the nucleus (blue). (F) The level of p-RyR2 and p-PLN were downregulated by miR-17-3p mimic or upregulated by inhibitor. (G) The peak FI of fluo-3 decreased in miR-17-3p transfected NMCMs and increased in inhibitor transfected NMCMs. ** p < 0.01, n = 15. (H) The FDHM of NMCMs activated by carbamylcholine was not affected by miR-17-3p or inhibitor. NS = not significant, n = 15. (I) The miR-17-3p can decrease the FI of 530nm and increase the 475nm in NMCMs; Inhibitor can increase the FI of 530 and decrease the 475nm. * p < 0.05, n = 15. (J) Both miR-17-3p and inhibitor had no effects on PLN and RyR2 in genetic level. NS = not significant.

Article Snippet: Anti-RyR2, anti-PLN, anti-p-PLN, anti-p-RyR2, anti-His were purchased from Abcam (Britain).

Techniques: Quantitative RT-PCR, Binding Assay, Luciferase, Reporter Gene Assay, Fluorescence, Transfection

Journal: International Journal of Biological Sciences

Article Title: Circ-HIPK3 Strengthens the Effects of Adrenaline in Heart Failure by MiR-17-3p - ADCY6 Axis

doi: 10.7150/ijbs.36149

Figure Lengend Snippet: Verification of existence of circ-HIPK3-miR-17-3p-ADCY6 axis. (A) Left: Bar graph showing that miR-17-3p could significantly decrease the fluorescence density of NMCMs with pmirGLO-wt-ADCY6-3'UTR; Right: The binding sites of WT and MUT sequence in ADCY6 3'UTR with miR-17-3p. * p < 0.05. (B) WB showing that miR-17-3p can decrease the level of ACDY6 and inhibitor can increase it. (C) ADCY6 can be up- or down-regulated by pCMV-ADCY6 or si-ADCY6 at genetic and protein level. * p < 0.05. (D) The overexpression of ADCY6 can increase the level of p-RyR2 and p-PLN and vice versa. (E) The ADCY6 overexpression can increase the peak FI of fluo-3 in NMCMs activated by carbamylcholine and vice versa. * p < 0.05, n = 15. (F) Bar graph showing the variation of ADCY6 in NMCMs had little effects on FDHM of Ca 2+ transient. NS = not significant, n = 15. (G) Left: The peak FI of 475nm can be downregulated by ADCY6 overexpression and upregulated by its under-expression; Right: The peak FI of 530nm can be upregulated by ADCY6 overexpression and downregulated by its under-expression. * p < 0.05, n = 15. (H) WB showing the increase of ADCY6 caused by circ-HIPK3 can be attenuated by miR-17-3p and the reduction induced by si-circ-HIPK3 can be rescued by inhibitor.

Article Snippet: Anti-RyR2, anti-PLN, anti-p-PLN, anti-p-RyR2, anti-His were purchased from Abcam (Britain).

Techniques: Fluorescence, Binding Assay, Sequencing, Over Expression, Expressing

Journal: International Journal of Biological Sciences

Article Title: Circ-HIPK3 Strengthens the Effects of Adrenaline in Heart Failure by MiR-17-3p - ADCY6 Axis

doi: 10.7150/ijbs.36149

Figure Lengend Snippet: AAV9-shRNA in vivo improved the cardiac function post MI. (A) qRT-PCR showed HIPK3 and circ-HIPK3 increased in NC group and decreased in experiment group respectively. * p<0.05. (B) Left: Representative images of hearts sections by TTC staining (Viable myocardium stained red, and the infarcted areas appeared pale). Dotted box showing the infarcted areas. Right: Bar graph showing percent of infarcted LV in experiment group decreased significantly compared to that in NC group. * p<0.001. (C)(D) Results of echocardiography showed that the cardiac function of heart in experiment group increased significantly compared with that in NC group. * p<0.05. (E) Upper: Masson staining showed the degree of fibrosis of heart in NC group was much higher than that in experiment group, normal group or control group. Lower: WB showed col1 (collagen 1) and col3 (collagen 3) increased significantly in NC group. (F) Immuno-blotting showing that ADCY6 increased in NC group but the level of RyR2, PLN and their phosphorylated form decreased in NC group. (G) QRT-PCR showed that PLN, RyR2 and SERCA2a decreased significantly in NC group when compared to these in experiment group but ADCY6 changed little. * p<0.05, NS = not significant.

Article Snippet: Anti-RyR2, anti-PLN, anti-p-PLN, anti-p-RyR2, anti-His were purchased from Abcam (Britain).

Techniques: shRNA, In Vivo, Quantitative RT-PCR, Staining

Journal: International Journal of Biological Sciences

Article Title: Circ-HIPK3 Strengthens the Effects of Adrenaline in Heart Failure by MiR-17-3p - ADCY6 Axis

doi: 10.7150/ijbs.36149

Figure Lengend Snippet: MiR-17-3p was the downstream target of circ-HIPK3. (A) Results of WB showing that p-RyR2 and p-PLN increased in circ-HIPK3 overexpressed NMCMs and vice versa. (B) QRT-PCR showing that PLN and RyR2 changed little in circ-HIPK3 over- or under- expressed NMCMs. NS = not significant. (C) Schematic image showing that seven possible binding sites of miR-17-3p to circ-HIPK3 were found. (D) Dual-luciferase reporter gene assay showed miR-17-3p could decrease the fluorescence density of NMCMs transfected with pmirGLO- wt -circ-HIPK3. * p<0.05 . (E) FISH assay showed circ-HIPK3 (red) can interact with miR-17-3p (green), which mainly were around the nucleus (blue). (F) The level of p-RyR2 and p-PLN were downregulated by miR-17-3p mimic or upregulated by inhibitor. (G) The peak FI of fluo-3 decreased in miR-17-3p transfected NMCMs and increased in inhibitor transfected NMCMs. ** p < 0.01, n = 15. (H) The FDHM of NMCMs activated by carbamylcholine was not affected by miR-17-3p or inhibitor. NS = not significant, n = 15. (I) The miR-17-3p can decrease the FI of 530nm and increase the 475nm in NMCMs; Inhibitor can increase the FI of 530 and decrease the 475nm. * p < 0.05, n = 15. (J) Both miR-17-3p and inhibitor had no effects on PLN and RyR2 in genetic level. NS = not significant.

Article Snippet: Anti-RyR2, anti-PLN, anti-p-PLN, anti-p-RyR2, anti-His were purchased from Abcam (Britain).

Techniques: Quantitative RT-PCR, Binding Assay, Luciferase, Reporter Gene Assay, Fluorescence, Transfection

Journal: International Journal of Biological Sciences

Article Title: Circ-HIPK3 Strengthens the Effects of Adrenaline in Heart Failure by MiR-17-3p - ADCY6 Axis

doi: 10.7150/ijbs.36149

Figure Lengend Snippet: Verification of existence of circ-HIPK3-miR-17-3p-ADCY6 axis. (A) Left: Bar graph showing that miR-17-3p could significantly decrease the fluorescence density of NMCMs with pmirGLO-wt-ADCY6-3'UTR; Right: The binding sites of WT and MUT sequence in ADCY6 3'UTR with miR-17-3p. * p < 0.05. (B) WB showing that miR-17-3p can decrease the level of ACDY6 and inhibitor can increase it. (C) ADCY6 can be up- or down-regulated by pCMV-ADCY6 or si-ADCY6 at genetic and protein level. * p < 0.05. (D) The overexpression of ADCY6 can increase the level of p-RyR2 and p-PLN and vice versa. (E) The ADCY6 overexpression can increase the peak FI of fluo-3 in NMCMs activated by carbamylcholine and vice versa. * p < 0.05, n = 15. (F) Bar graph showing the variation of ADCY6 in NMCMs had little effects on FDHM of Ca 2+ transient. NS = not significant, n = 15. (G) Left: The peak FI of 475nm can be downregulated by ADCY6 overexpression and upregulated by its under-expression; Right: The peak FI of 530nm can be upregulated by ADCY6 overexpression and downregulated by its under-expression. * p < 0.05, n = 15. (H) WB showing the increase of ADCY6 caused by circ-HIPK3 can be attenuated by miR-17-3p and the reduction induced by si-circ-HIPK3 can be rescued by inhibitor.

Article Snippet: Anti-RyR2, anti-PLN, anti-p-PLN, anti-p-RyR2, anti-His were purchased from Abcam (Britain).

Techniques: Fluorescence, Binding Assay, Sequencing, Over Expression, Expressing

Journal: International Journal of Biological Sciences

Article Title: Circ-HIPK3 Strengthens the Effects of Adrenaline in Heart Failure by MiR-17-3p - ADCY6 Axis

doi: 10.7150/ijbs.36149

Figure Lengend Snippet: AAV9-shRNA in vivo improved the cardiac function post MI. (A) qRT-PCR showed HIPK3 and circ-HIPK3 increased in NC group and decreased in experiment group respectively. * p<0.05. (B) Left: Representative images of hearts sections by TTC staining (Viable myocardium stained red, and the infarcted areas appeared pale). Dotted box showing the infarcted areas. Right: Bar graph showing percent of infarcted LV in experiment group decreased significantly compared to that in NC group. * p<0.001. (C)(D) Results of echocardiography showed that the cardiac function of heart in experiment group increased significantly compared with that in NC group. * p<0.05. (E) Upper: Masson staining showed the degree of fibrosis of heart in NC group was much higher than that in experiment group, normal group or control group. Lower: WB showed col1 (collagen 1) and col3 (collagen 3) increased significantly in NC group. (F) Immuno-blotting showing that ADCY6 increased in NC group but the level of RyR2, PLN and their phosphorylated form decreased in NC group. (G) QRT-PCR showed that PLN, RyR2 and SERCA2a decreased significantly in NC group when compared to these in experiment group but ADCY6 changed little. * p<0.05, NS = not significant.

Article Snippet: Anti-RyR2, anti-PLN, anti-p-PLN, anti-p-RyR2, anti-His were purchased from Abcam (Britain).

Techniques: shRNA, In Vivo, Quantitative RT-PCR, Staining

Journal: PLoS ONE

Article Title: Heart Failure-Inducible Gene Therapy Targeting Protein Phosphatase 1 Prevents Progressive Left Ventricular Remodeling

doi: 10.1371/journal.pone.0035875

Figure Lengend Snippet: A : Immunoblots of the key SR phosphoproteins and analysis of the phosphorylation levels by using phosphospecific antibodies in LV homogenates at 3 months after gene transfer. B : Summaries of the phospholylation levels of PLN at Ser16 and RyR at Ser2808. “*” indicates p<0.05 vs. the NCshRNA treated group. (n = 8 in PP1βshRNA treated group and n = 8 in NCshRNA treated group). C : Expression analysis of normalized BNP (nBNP) using real-time RT-PCR from the AAV9 shRNA transfected heart tissue. “*” indicates p<0.05 vs. the NCshRNA treated group. (n = 8 in the NCshRNA treated group, n = 8 in the PP1βshRNA treated group). D : Representative images of Heidenhain's trichrome staining in the AAV9-BNP-EmGFP-NCshRNA- andAAV9-BNP-EmGFP-PP1βshRNA treated hearts at 3 months after gene transfer. The lower graph shows the quantitative image analysis of percentage of the area of interstitial fibrosis. “*” indicates p<0.05 vs. the NCshRNA treated group. (n = 6 in NCshRNA treated group and n = 7 in PP1βshRNA treated group).

Article Snippet: The following antibodies were obtained from commercially available sources: the antibodies for PP1β (ab16369, ab53315), and PLN (ab2865, clone 2D12: Abcam, Cambridge, UK); phosphorylated-PLN at Ser16, PLN, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Chemicon International), phosphorylated-PLN at Thr17, and phoshphorylated-RyR2 at Ser2808 (Badrilla, Leeds, UK); RyR2 (clone C3–33: Sigma-Aldrich, St. Louis, MO), SERCA2a (clone N-19: Santa Cruz Biotechnology, Santa Cruz, CA), cardiac troponin I (cTn-I: clone 19C7), phospho-TnI at Ser22,23 (clone 5E6: Genetex, San Antonio, TX), and GFP(ab290: Abcam).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, shRNA, Transfection, Staining

Journal: PLoS ONE

Article Title: Preserved Cardiac Function despite Marked Impairment of cAMP Generation

doi: 10.1371/journal.pone.0072151

Figure Lengend Snippet: A . PKA activity in isolated cardiac myocytes without stimulation (Basal) or stimulated with isoproterenol (Iso; 10 µM, 10 min) or NKH477 (NKH; 10 µM, 10 min). AC6mut expression reduced basal PKA activity (p = 0.01) and both Iso (p = 0.001) and NKH (p = 0.001) activities were reduced as well (n = 3, each group). B . The expression of key signaling proteins and their phosphorylation are shown in immunoblots using left ventricular homogenates from AC6mut and control mice. No group differences were observed. Shown are PKA catalytic unit, phospho (P) and Total (T) PKA regulatory subunits II-α and II-β, PKCα, phosphodiesterase type 3A (PDE3A), phospho-troponin I (P22/23-TnI), and total TnI. C . Phosphorylation of RyR2, PLB and TnI before and after isoproterenol stimulation was assessed in cardiac myocytes isolated from each group. Basal phosphorylation of RyR2, PLB and TnI showed no group differences. Isoproterenol stimulation was associated with increased phosporylation of RyR2, PLB, and TnI in both groups, but was more extensive in cardiac mycoytes from AC6mut mice. D . The data from Fig. 2C indicating that isoproterenol stimulation was associated with increased phosporylation of RyR2, PLB, and TnI in cardiac mycoytes from AC6mut mice are shown in graphic format, normalized for loading (GAPDH). The increase in TnI phosphorylation was not statistically significant (p = 0.07).

Article Snippet: Additional antibodies used included: calreticulin (ABR Affinity, 1∶1,000); calsequestrin (Novus Biologicals, 1∶1,000); GAPDH (Fitzgerald, 1∶20,000); PDE3A (Santa Cruz, 1∶500); PKA catalytic subunit (BD Transduction, 1∶1,000); p-PKA catalytic subunit (Cell Signaling, 1∶1,000); PKA-RIIα and PKA-RIIβ (BD Transduction, 1∶1,000); phospho-PKA-RIIα (S96) and phospho-PKA-RIIβ (S114) (Santa Cruz, 1∶200); PKCα catalytic subunit (Santa Cruz, 1∶200); PLB (Affinity Bioreagents, 1∶5,000); phospho S16-PLB (Badrilla, 1∶3,000 dilution); phospho-RyR2 (S2808) (Abcam, 1∶1,000); S100A1 (Epiyomics, 1∶1,000); SERCA2a (Enzo, 1∶1,000); troponin I and phospho-TnI (S22/23) (Cell Signaling, 1∶1,000 each).

Techniques: Activity Assay, Isolation, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Increased Muscle Stress-Sensitivity Induced by Selenoprotein N Inactivation in Mouse: A Mammalian Model for SEPN1 -Related Myopathy

doi: 10.1371/journal.pone.0023094

Figure Lengend Snippet: A ) Electron microscopy image of gastrocnemius muscles from 6 month-old wild-type and mutant mice. No defect in sarcomere organization, mitochondria morphology (arrows) and triads structure (brackets) were observed in mutant (right panel), compared to wild-types (left panel). Scale bars, 1 µm (top) and 100 nm (bottom). B ) Immunostaining for DHPR (green) and RyR1 or RyR3 (red) on longitudinal sections of quadriceps from 2 month-old mice, revealing normal localization of both receptors in mutant mice compared to controls. Nuclei are revealed by DAPI staining. Boxed regions in the merged images are magnified as inserts (right) showing intracellular co-localization (yellow). Scale bar is 10 µm. C ) Skeletal muscle microsomes extracts from Sepn1 +/+ or Sepn1 −/− littermates were immunoblotted for Ryanodine Receptor type 1 (RyR1), Ryanodine Receptor type 3 (RyR3), Dihydropyridine Receptor (DHPR), Selenoprotein N (SelN) and triadin isoform Trisk 95 (Trisk 95).

Article Snippet: The following antibodies were used on 8 µm muscle cryosections: embryonic MHC (DSHB, F1.652), slow MHC (DSHB, A4.840), fast MHC (DSHB, A4.74), DHPR, RyR3 (Chemicon, MAB427 and AB9082, respectively), laminin (Abcam, ab11575), RyR1 and Trisk95 antibodies (kind gift from Dr. Isabelle Marty, Grenoble Institut des Neurosciences, France).

Techniques: Electron Microscopy, Mutagenesis, Immunostaining, Staining

Journal: PLoS ONE

Article Title: Increased Muscle Stress-Sensitivity Induced by Selenoprotein N Inactivation in Mouse: A Mammalian Model for SEPN1 -Related Myopathy

doi: 10.1371/journal.pone.0023094

Figure Lengend Snippet: A ) Electron microscopy image of gastrocnemius muscles from 6 month-old wild-type and mutant mice. No defect in sarcomere organization, mitochondria morphology (arrows) and triads structure (brackets) were observed in mutant (right panel), compared to wild-types (left panel). Scale bars, 1 µm (top) and 100 nm (bottom). B ) Immunostaining for DHPR (green) and RyR1 or RyR3 (red) on longitudinal sections of quadriceps from 2 month-old mice, revealing normal localization of both receptors in mutant mice compared to controls. Nuclei are revealed by DAPI staining. Boxed regions in the merged images are magnified as inserts (right) showing intracellular co-localization (yellow). Scale bar is 10 µm. C ) Skeletal muscle microsomes extracts from Sepn1 +/+ or Sepn1 −/− littermates were immunoblotted for Ryanodine Receptor type 1 (RyR1), Ryanodine Receptor type 3 (RyR3), Dihydropyridine Receptor (DHPR), Selenoprotein N (SelN) and triadin isoform Trisk 95 (Trisk 95).

Article Snippet: The following antibodies were used on 8 µm muscle cryosections: embryonic MHC (DSHB, F1.652), slow MHC (DSHB, A4.840), fast MHC (DSHB, A4.74), DHPR, RyR3 (Chemicon, MAB427 and AB9082, respectively), laminin (Abcam, ab11575), RyR1 and Trisk95 antibodies (kind gift from Dr. Isabelle Marty, Grenoble Institut des Neurosciences, France).

Techniques: Electron Microscopy, Mutagenesis, Immunostaining, Staining

Journal: Mediators of Inflammation

Article Title: Toll-Like Receptor-Mediated Cardiac Injury during Experimental Sepsis

doi: 10.1155/2020/6051983

Figure Lengend Snippet: Effects of LPS on murine HL-cells. Fluorescence intensity of troponin I expression of HL-1 cells (a). Troponin I elevation in supernatant fluids from HL-1 cells (b) and from human cardiomyocytes (c). mRNA expression of pyrogenic receptor (P2X7) in human cardiomyocytes (d). HL-1 cells were treated with 20 μ g/ml LPS and human cardiomyocytes (iPS) with 10 μ g/ml LPS for 6 h (black bars). Additional treatment included treatment with 20 μ g/ml or 10 μ g/ml LPS for 5 h, respectively, and for one further hour with either 1 mM ATP or 1 μ M nigericin (grey bars). Control groups were incubated in a cell culture medium without any supplements for 6 h (white bars). Amount of cellular reactive oxygen species (ROS) (e). Fluorescence intensity of ryanodine receptor 1 (RyR1) (f). n = 6 per group; ∗ p < 0.05. All values are expressed as mean ± SEM.

Article Snippet: Unspecific binding sites were blocked with 10% goat serum, and specific antigen detection was performed by incubating cells with the respective primary antibodies for ryanodine receptor 1 (RyR1) (Abcam, Cambridge, UK), desmin (GeneTex, Irvine, CA, USA), and troponin I (Abcam, Cambridge, UK) for overnight at 4°C.

Techniques: Fluorescence, Expressing, Incubation, Cell Culture

Journal: PLoS ONE

Article Title: Increased Na + /Ca 2+ Exchanger Expression/Activity Constitutes a Point of Inflection in the Progression to Heart Failure of Hypertensive Rats

doi: 10.1371/journal.pone.0096400

Figure Lengend Snippet: A) Representative immunoblots and average results of the expression and/or phosphorylation of B) NCX; C) SERCA2a; D) PLN; E) SERCA2a/PLN ratio; F) NCX/SERCA2a ratio; G) pCaMKII; H) pThr 17 -PLN; I) pSer 2814 -RyR2; J) pSer 16 -PLN; K) pSer 2808 -RyR2. The results are expressed as percentage of values obtained in W of the same age. Protein levels were normalized to the loading control glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Phosphorylation of PLN and RyR2 was expressed as ratio between phosphorylated and non-phosphorylated forms of the proteins. While SERCA2a expression showed no differences at any time point studied, the expression of NCX was significantly higher only in SHRF. In this latter group, PLN expression and SERCA2a/PLN ratio did not change with respect to W, therefore the ratio NCX/SERCA2a was significantly enhanced. CaMKII and Thr 17 -PLN phosphorylations significantly increased from 3 mo in SHR with respect to W. PKA-dependent Ser 16 phosphorylation of PLN increased at 3 mo and then decreased. Phosphorylation of Ser 2808 and Ser 2814 of RyR2 did not change at any age studied. *p<0.05 with respect to W of the same age; n≥4 animals per group.

Article Snippet: Blots were probed overnight with the following antibodies: Bcl-2 1∶1000 (Santa Cruz biotechnology, Santa Cruz, CA, USA), Bax 1∶1000 (Santa Cruz biotechnology, Santa Cruz, CA, USA), Sarcoplasmic Reticulum Ca 2+ -ATPase (SERCA2a) 1∶1000 (Thermo Scientific, Rockford, IL, USA), Na + /Ca 2+ exchanger (NCX) 1∶1000 (Millipore, Billerica, MA, USA), phospholamban (PLN) 1∶1000 (ABR, Golden, CO, USA), Thr 17 and Ser 16 -phosphorylated PLN 1∶1000 (pThr 17 and pSer 16 , respectively) (1∶5000 (Badrilla, Leeds, UK), Ryanodine Receptors (RyR2) 1∶1000 (ABR, Golden, CO, USA), Ser 2814 and Ser 2809 -phosphorylated RyR2 1∶1000 (Badrilla, Leeds, UK), pCaMKII 1∶1000 (Abcam, Cambridge, MA, USA).

Techniques: Western Blot, Expressing