The rule both stores actually apply
Apple’s screenshot specification is one sentence long on this point: images can’t include alpha channels or transparencies. Google’s asset requirements say the same thing in the format itself — screenshots and the feature graphic must be JPEG or 24-bit PNG with no alpha.
Read that carefully, because it is the part that costs people an afternoon: 24-bit means three channels. A PNG with four channels is a 32-bit file, and it is refused whether or not a single pixel in it is see-through. The store does not say which file was wrong or why — the upload just fails validation.
Why re-saving it somewhere else does not fix it
Nearly every browser-based image tool re-encodes through an HTML canvas, and browsers always write canvas PNGs as 32-bit RGBA no matter what was drawn on them. So the file comes back flattened, visually correct, and still carrying the channel that got it rejected in the first place.
This page writes the PNG byte by byte instead: colour type 2, eight bits per channel, filtered and deflated the way any encoder would, with no alpha channel in the file to begin with. The transparent pixels are composited onto the background colour you choose before encoding, so what you see is what gets written.
The one asset that must keep its alpha
Google Play’s 512 × 512 app icon is the exception, and it goes the other way: it must be a 32-bit PNG with alpha, under 1024 KB. Strip that one and the Play Console will refuse it. Everything else — every screenshot, the 1024 × 500 feature graphic, and every App Store image — must have none.