## 3 Results
### PARP7 represses ER\(\alpha\) Activity
A comparison between our lab-generated antibody and a commonly used commercially available anti-PARP7 antibody confirmed its increased selectivity for PARP7 (Supplementary Figure S1B,C). Our lab generated antibody was raised against murine Parp7, but it also cross-reacts with human PARP7, albeit with reduced sensitivity. In E2 treated MCF-7 cells, we were unable to detect increased PARP7 protein levels at either timepoint (Figure 2E). This was most likely due to rapid turnover or instability of PARP7 [32] and a low sensitivity of anti-PARP7 antibody to detect human PARP7. However, PARP7 protein levels were increased in cells co-treated with E2+RBN-2397 for 4 or 24 h compared with RBN-2397 alone. This indicated that PARP7 protein expression is induced by E2, but that the inhibition PARP7 catalytic activity was necessary  
Figure 2: Inhibition of PARP7 activity stabilizes PARP7 protein levels and increases ER\(\alpha\) activity. (**A**) RBN-2397 stabilizes PARP7 protein levels and decreases catalytic activity. COS-1 cells were transfected with GFP-PARP7 and treated with 0.1% DMSO or 100 nM RBN-2397 for 24 h. Samples were immunoprecipitated with anti-GFP, and membranes were blotted with anti-GFP and anti-ADP-ribose antibodies. (**B**) COS-1 cells were transfected with GFP-PARP7 or GFP-PARP7H532A and treated with 0.1% DMSO or 100 nM RBN-2397 for 24 h. (**C**) _Parp7\({}^{\prime+\prime}\), Parp7\({}^{\prime-\prime}\)_ and _or_ _Parp7\({}^{\prime+532A}\)_ MEFs were treated with 0.1% DMSO or 100 nM RBN-2397 for 24 h. The membrane was probed with our lab generated anti-PARP7. (**D**) Treatment with RBN-2397 increases mRNA expression of ER\(\alpha\) target gene GREB1. Wildtype MCF-7 cells were treated with 0.1% DMSO, 10 nM E2 or co-treated with E2 and 100 nM RBN-2397 for 24 h. The asterisk * denotes significant differences (\(p<0.05\)) from DMSO, and the hash mark # denotes significant differences (\(p<0.05\)) compared to E2 treatment alone. (**E**) E2 stimulation increases PARP7 protein expression. MCF-7 cells were treated with 10 nM E2 for 0, 4 and 24 h, together with control (no treatment) or 24 h treatment with 100 nM RBN-2397. The membrane was blotted with our lab generated anti-PARP7, anti-ER\(\alpha\), or anti-PARP7 (Abcam; ab84664) antibodies. PARP7 bands are visible in samples co-treated with E2 and RBN-2397. Anti-PARP7 (ab84664), did not detect endogenous PARP7, but rather detected a protein at approximately 100 kDa in all lanes. to stabilize PARP7 protein levels to detect the protein with our antibody. The detected band was slightly higher than PARP7's predicted 76 kDa molecular weight, but similar to that observed in MEFs (Figure 2C and Supplementary Figure S1C). The findings, however, support previous studies of transfected full length and truncated PARP7 that show that it runs higher than its predicted weight [17]. A commercially available anti-PARP7 (a84664) failed to detect PARP7 after co-treatment with E2 and RBN-2397. A strong band at approximately 100 kDa was detected in all lanes. Interestingly, E2-dependent decreases in ER\(\alpha\) protein levels were reduced upon PARP7 inhibition, suggesting that PARP7 regulates ER\(\alpha\) proteolytic degradation.