Will my GPU lose x16 support if second PCIe slot is used for

pcie x16 expansion slot
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There are a number of simple ways to check for a PCIe x16 slot, though some may require you to open the machine up So you'll need some

Card capacity and size Mac Pro has eight PCIe x16* sized slots that accept many different types of PCIe cards: Slot 8 is x4 in size Mac Pro Expansion

pcie4 slot In ASUS 「PCIe x16 Slots - 7」

pcie 30 in 40 slot expansion cards, allowing you to install a PCIe card in an x1 slot The PCI Express x1 to x16 adapter accommodates low profile x2, x4, x8, x12 or x16